Pharmaceutical dipeptide compositions and methods of use thereof: immunodepressants

ABSTRACT

Methods of treatment of subjects for decreasing cell mediated autoimmunity or humoral autoimmunity by administering an R&#39;-Glu-Trp-R&#34; pharmaceutical preparation useful in subjects having autoimmune diseases.

This application is a continuation-in-part of Ser. No. 08/337,341 filedNov. 10, 1994 now U.S. Pat. No. 5,538,951, and a continuation-in-part ofSer. No. 08/278,463 filed Jul. 21, 1994 (abandoned), said Ser. No.08/337,341 filed Nov. 10, 1994 which is a continuation-in-part of Ser.No. 08/257,495 filed Jun. 7, 1994 (abandoned), which is a continuationof Ser. No. 07/783,518 filed Oct. 28, 1991 (abandoned), which is acontinuation-in-part of Ser. No. 07/678,129 filed Apr. 1, 1991(abandoned), which is a continuation-in-part of Ser. No. 07/415.283filed Aug. 30, 1989 (abandoned), which is a 371 of PCT/SU88/00255 filedDec. 14, 1988. All the above patent applications are hereby incorporatedby reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates generally to pharmaceutical compositionscontaining peptides having immunomodulating properties and moreparticularly to pharmaceutical compositions of tryptophan-containingdipeptides and methods of use thereof.

BACKGROUND OF THE INVENTION

The lymphoid system performs critical functions in animals and man thatinclude preventing and combating infection, and surveillance and immuneelimination of tumor cells. Loss of immune function leads to animmunocompromised status that can predispose the host to serious andlife-threatening disease. Functional abnormalities may be present in anyof the elements that participate in mediating an immune response, e.g.,cellular or humoral elements such as granulocytes, lymphocytes,complement, antibody, or cytokines.

Immune deficiency may result from many different etiologies includinghereditary genetic abnormalities (e.g., Chediak-Higashi Syndrome, severecombined immunodeficiency, chronic granulomatous disease, DiGeorgesyndrome) exposure to radiation, chemotherapy, heavy metals orinsecticides; or, acquired as a result of bacterial, viral, parasitic orfungal infection.

The immune system is normally regulated by cellular and soluble elementsand loss of regulatory control may result in autoimmune disease, e.g.,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,Sjogren's syndrome, and insulin-dependent type I diabetes. Conventionalimmunosuppressive drugs that are used to treat autoimmune diseases havegeneralized and non-specific effects on the immune system that canpredispose infection and increase the risk of malignancy. Methods tocompensate for decreased innate resistance to infection followingradiation orchemotherapy are therefore of great therapeutic value.

Thymus tissue extracts affecting proliferation and/or differentiation ofT-lymphocytes have been reported, including, e.g., "thymosine" (1),"Thymaline" (2), "T-activine" (3), "thymosin-α₁ (4), "thymosin fraction5" (a heat-stable fraction isolated from calf thymus extracts), andothers. Commonly tissue extracts exist as complex mixtures that includepolypeptides, e.g., several peptides have been isolated from thymosinfraction 5, such as thymosin alpha₁ (28 amino acids, U.S. Pat. No.4,079,127), thymosin beta₄ (44 amino acids, Low, el al., Proc. Nat'lAcad. Sci. USA 78:1162-1166 (1981)), thymosin beta₈ (39 amino acids,U.S. Pat. No. 4,389,343) and thymosin beta₉ (41 amino acids, U.S. Pat.No. 4,389,343). Thymosin-α₁ fragments and dimers have also beendescribed in U.S. Pat. Nos. 4,396,605; 4,470,926; 4,612,365; and4,910,296.

Widespread use of purified tissue extract fractions in medical practicehas been hindered by variability of preparations, low yields, lack ofhighly purified and characterized reproducible sources, and problemsassociated with preparing complex mixtures having standardizedbiological potency. In addition, complexity and variability of tissueextracts potentially affect stability, toxicity, and safety.Alternatively, synthesis of large biologically active polypeptides is atpresent difficult and expensive particularly under manufacturingconditions required for pharamaceutical preparations.

Alternatively, synthesis of large biologically active polypeptides is atpresent difficult and expensive, particularly under the manufacturingconditions required for pharmaceutical preparations.

T-lymphocytes participate in cellular and humoral immune responsestriggered by binding of foreign molecules to lymphocyte cell surfacereceptors. The erythrocyte rosette receptor defined by the CD2 cellfunctions as both an intercellular adhesion molecule and a cell surfacesignal transduction molecule. CD2 reportedly binds PHA and is involvedin PHA-mediated lymphocyte blastogenesis. Binding of anti-CD2 antibodiesto the CD2 receptor is also reportedly able to trigger T-lymphocyteblastogenesis and this pathway of activation may be independent of theCD3/T-cell receptor complex. T-cell CD2 binding to LFA-3/CD58 maymediate intercellular adhesive binding of T-lymphocytes to B-lymphocytesand thymic epithelial cells. CD2 binding to CD59 and CD48 ligands mayfacilitate intercellular binding to other cell types. Lymphocyte cellsurface CD4 and CD8 molecules define MHC class specificity of T-helper,T-suppressor and cytotoxic T-lymphocytes. (Paul, W. E. Ed. 1993."Fundamental Immunology, 3rd. Edition", Raven Press, N.Y. pp.541-5.)

SUMMARY OF THE INVENTION

Methods have been discovered for treating immunocompromised subjects toincrease one or more indicia of cell mediated immunity (CMI), humoralimmunity, or innate resistance to infection, by administeringpharmaceutical preparations of R'-Glu-Trp-R". The results of in vitrostudies showed that L-Glu-L-Trp dipeptide increased expression ofaccessory molecules on the surface of thymocytes and matureT-lymphocytes as evidenced by i) increased E-rosette forming cells(E-RFC) in thymocyte cultures after incubation with dipeptide; ii)increased E-RFC in cultures of thymocytes from aged animals afterincubation with dipeptide; and, iii) increased expression of OKT 4⁺ incultures of human peripheral blood T-lymphocytes from patients withsecondary immunodeficiency syndromes following incubation withL-Glu-L-Trp. L-Glu-L-Trp dipeptide did not measurably upregulated CD8expression on lymphocytes. Increased expression of CD2 and CD4 accessorymolecules on T-lymphocytes is compatible with a heighten the state ofinnate or induced immunity to infection, e.g., by upregulating T-helperand cytotoxic T-lymphocytes to respond to lower levels of antigen. Totest this hypothesis, in vivo studies were conducted in which theimmunological effects of L-Glu-L-Trp dipeptide were tested inexperimental animal models. L-Glu-L-Trp treatments mobilized and alteredtissue distribution of lymphocytes in experimental animals, activatedmonocytes and increased phagocytic activity of granulocytes. Animalmodel studies in mice showed that treatments with L-Glu-L-Trp decreasedincidence of mortality from acute bacterial infection with E. coli,Pseudomonas aeruginosa, and staphylococci. In irradiated guinea pigs and5-fluorouracil (5-FU) immunosuppressed mice, L-Glu-L-Trp treatmentsincreased the number of lymphocytes and T-lymphocytes in peripheralblood. When injected locally, L-Glu-L-Trp increased the activation stateof resident tissue macrophages (as measured by NBT reduction); and,promoted neutrophil infiltration into the tissue in response to asterile inflammatory mediator (proteose peptone). L-Glu-L-Trp treatmentsincreased in vitro expression of CD4 (but not CD8) on lymphocytesisolated from patients with secondary immunodeficiency syndromes.Clinical studies showed increased indicia of CMI, humoral immunity orinnate immunity in the following patients treated with L-Glu-L-Trppharmaceutical preparations: namely, patients with acute and chronicinfections including respiratory infections, pleuritis, pelvicinflammatory diseases, infections of leprosy, tuberculosis,staphylococcal pyoderma, Dengue fever, chronic viral hepatitis, Shigelladysentery, malaria, influenza, and tuberculosis. L-Glu-L-Trp treatmentsalso i) alleviated certain clinical symptoms in patients with autoimmunedisease and allergy; ii) decreased complication rates and increasedlymphocyte counts in cancer patients following radiation therapy; iii)increased lymphocyte counts in individuals exposed to accidentalenvironmental radiation and surgical thymectomy; and, iii) increasedlymphocyte counts in patients with secondary immunodeficiency.L-Glu-L-Trp showed efficacy in both prophylactic and therapeuticprotocols. L-Glu-L-Trp treatments also proved useful for alleviatingcertain symptoms of systemic toxicity in patients with acute bacterial,viral, and parasitic infections.

HIV-infected patients are one group of immunocompromised subjects thatmay benefit from treatments with R'-Glu-Trp-R", however, it isunderstood that such treatments are not intended as a cure for AIDS orARC, but rather for possible use in treating complications ofHIV-infection, e.g., opportunistic bacterial and viral infections.

Embodiments of the invention provide methods of treatment usingR'-Glu-Trp-R" pharmaceutical preparations to induce a heightened stateof cell mediated immunity, humoral immunity, or innate resistance toinfection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically depicts the results of studies in experimentalanimals designed to test an L-Glu-L-Trp prophylactic regimen fortreating an acute gram negative bacterial infection, as described inEXAMPLE 32. L-Glu-L-Trp was administered intraperitoneally (ip) on adaily basis from day -3 to day +1 at dosages that spanned 5 logs ofconcentration (i.e., 0.01 μg/kg to 100 jig/kg). The bacterial challengewas administered on day 0. For combination treatments with antibiotics,the same five daily ip doses of L-Glu-L-Trp were administered along withfive daily ip doses of the indicated antibiotics.

FIG. 1A graphically depicts the results of the prophylactic L-Glu-L-Trptreatment, i.e., expressed as the percentage of mice surviving (%) aperitoneal infection at 24, 48, or 72 hours after injection of an LD₁₀₀dose of E. coli. Mice in the experimental group received the L-Glu-L-Trptreatment, while those in the control group did not.

FIG. 1B graphically depicts the results of the prophylactic combinationL-Glu-L-Trp and antibiotic treatment, i.e., expressed as the percentagemice surviving (%) an ip LD₁₀₀ challenge of E. coil at 24, 48, or 72hours. Mice in the experimental group received the prophylactic courseof combination therapy with L-Glu-L-Trp and ampicillin; mice in thecontrol group were untreated; and, mice in the antibiotic control groupwere treated with only ampicillin (amp).

FIG. 1C graphically depicts the data presented in FIG. 1A, but plottedin a dose-response type fashion.

FIG. 1D graphically depicts the data presented in FIG. 1B, but plottedin a dose-response type fashion.

FIG. 2 graphically depicts the results of studies parallel to thosedescribed above in regard to "FIG. 1", but using Pseudomonas aeruginosaas the gram negative bacterial pathogen instead of E. coli, i.e., asdescribed in EXAMPLE 33.

FIG. 2A graphically depicts the results of studies parallel to thosedescribed in regard to FIG. 1A, above, but using Pseudomonas aeruginosa.Mice in the experimental group received a prophylactic course ofL-Glu-L-Trp treatment, while those in the control group did not.

FIG. 2B graphically depicts the results of studies parallel to thosedescribed in regard to FIG. 1B, above, but using Pseudomonas aeruginosa.Mice in the experimental group received a prophylactic course ofcombination therapy using L-Glu-L-Trp and gentamicin; mice in thecontrol group were untreated; and, mice in the antibiotic control groupwere treated with only Gentamycingentamicin (gen).

FIG. 2C graphically depicts the data presented in FIG. 2A plotted in adose-response type fashion.

FIG. 2D graphically depicts the data presented in FIG. 2B plotted in adose-response type fashion. FIG. 3 graphically depicts the results ofstudies parallel to those described above in regard to "FIG. 1", butusing staphylococci as a gram positive bacterial pathogen instead of E.coli, i.e., as described in EXAMPLE 34, below.

FIG. 3A graphically depicts the results of studies parallel to thosedescribed in regard to FIG. 1A, above, but using staphylococci. Mice inthe experimental group received a prophylactic course of L-Glu-L-Trptreatment, while those in the control group did not.

FIG. 3B graphically depicts the results of studies parallel to thosedescribed in regard to FIG. 1B, above, but using staphylococci. Mice inthe experimental group received a prophylactic course of combination iptherapy using both L-Glu-L-Trp and ampicillin; mice in the control groupwere untreated; and, mice in the antibiotic control group were treatedip with only ampicillin (amp).

FIG. 3C graphically depicts data obtained in a parallel experiment tothat of

FIG. 3A, (but including additional dosages); plotted in a dose-responsetype fashion.

FIG. 3D graphically depicts data obtained in a parallel experiment tothat of

FIG. 3B, (but including additional dosages); plotted in a dose-responsetype fashion.

FIG. 3E graphically depicts data obtained in a dose-response experimentsimilar to that of FIG. 3C, but with L-Glu-L-Trp therapy administeredintramuscularly (im) route of injection rather than ip.

FIG. 3F graphically depicts data obtained in a dose-response experimentsimilar to that of FIG. 3D, but with combination therapy administered bythe im route rather than ip.

DETAILED DESCRIPTION OF THE INVENTION Introduction

Thymalin was disclosed in U.S. Pat. No. 5,070,076 as a mixture ofpolypeptides 80-90% of which have molecular sizes in the range of 600 to6000 daltons and isoelectric points in the range of 3.5-6.7. The totalmixture of polypeptides reportedly had a composition (molar percentage)as follows: Asp (5.8), Thr (5.7), Ser (5.7), Glu (6.2), Pro (10.1), Gly(8.6), Ala (10.7), Cys (3.4), Val (8.8), Met (trace), Ile (3.9), Leu(6.7), Tyr (2.4), Phe (3.2), His (2.9), and Lys (7.0). Isolation andpurification of the polypeptides led to eventual identification ofseveral biologically active peptides including one peptide having aGlu-Trp sequence. Synthetic L-Glu-L-Trp peptides were discovered toexhibit biological activities in vitro and in certain in vivo systems.The Pharmacologic Committee of the U.S.S.R. approved therapeutic use ofL-Glu-L-Trp synthetic peptides on Jun. 19, 1990. In considering possibleprior "use" and "art" issues it is valuable to consider the followingnoteworthy dates in opening-up of the former Soviet Socialist Republics:namely, Boris Yeltsin was elected President of the Russian Republic inJune of 1991; on Aug. 24, 1991 Gorbachev resigned as General Secretaryof the Communist Party and dissolved the Central Committee; and the lastmeeting of the Congress of Peoples Deputies took place in September of1991 (David Remnick, "Lenins Tomb", Vintage Books, Random House, Inc.,N.Y. 1994).

The results of studies disclosed in the Examples below show thatL-Glu-L-Trp upregulates CD2 expression on T-lymphocytes in vitro and invivo. Other studies show immunological effects of L-Glu-L-Trp onlymphocytes, mononuclear phagocytes, neutrophils, and on immunologicmediator pathways underlying expression of inflammation, allergy,endotoxic shock and cellular and humoral immunity.

As used herein the symbols for amino acids are according to theIUPAC-IUB recommendations published in Arch. Biochem. Biophys. 115:1-12(1966) with the following single letter symbols for the amino acids:

    __________________________________________________________________________    L, Leu, Leucine                                                                          V, Val, Valine                                                                        Y, Tyr, Tyrosine                                                                       D, Asp, Aspartic Acid                             I, Ile, Isoleucine                                                                       P, Pro, Proline                                                                       W, Trp, Tryptophan                                                                     E, Glu, Glutamic Acid                             M, Met, Methionine                                                                       G, Gly, Glycine                                                                       N, Asn, Asparagine                                                                     K, Lys, Lysine                                    T, Thr, Threonine                                                                        A, Ala, Alanine                                                                       Q, Gln, Glutamine                                                                      R, Arg, Arginine                                  F, Phe, Phenylalanine                                                                    S, Ser, Serine                                                                        C, Cys, Cysteine                                                                       H, His, Histidine                                 __________________________________________________________________________

The symbols for protective groups used in peptide synthesis aredescribed in Schrodex and Lubke, "The Peptides", Academic Press, N.Y.1965, e.g., Boc for t-butyloxycarbonyl and Bzl for benzyl. Otherabbreviations include: HPLC for high pressure liquid chromatography; TFAfor trifluoroacetic acid; K_(D) for dissociation constant; K_(a) forassociation constant; L_(eq) for equilibrium constant; f.a. for fattyacid; E for erythrocyte; E-RFC for E-rosette forming cell; EA forerythrocyte antibody; EAC for erythrocyte antibody complement; APC forantigen presenting cell; PHA for phytohemagglutinin; Con-A forConcanavalin-A; LPS for lipopolysaccharide; IL for interleukin; CSF forcolony stimulating factor; IFN for interferon; CTL for cytotoxicT-lymphocyte; NK-cell for natural killer cell; BM for bone marrow; PBLfor peripheral blood leukocyte; LN for lymph node; KLH for keyholelimpet hemocyanin; ELISA for enzyme linked immunosorbent assay; FIA forfluorescence immunoassay; TRF for time resolved florescence assay; RIAfor radioimmunoassay; AST for hepatic alanyl-seryl transaminase enzyme;ALT for alanyl-leucyl transaminase; ATIII for antithrombin III; PTT forprothrombin time; FDP for fibrin degradation products; ARVI for acuterespiratory viral infection; ip for intraperitoneal; im forintramuscular; sc for subcutaneous; id for intradermal; iv forintravenous; po for peros, i.e., oral; and, in for intranasal.

The present invention provides for a "Glu-Trp pharmaceuticalpreparation" comprising an R'-Glu-Trp-R" dipeptide, (e.g., L-Glu-L-Trp),or a cyclic form thereof according to Formula I, or a derivative oranalog thereof according to Formula II, or a multimeric form thereofaccording to Formula III, the latter including linear and branchedmultimers, cyclic forms, and cyclic polymers thereof R'-Glu-Trp-R" isalso referred to interchangeably as "EW", or "EW dipeptide," using thenormal convention wherein the first named amino acid is the aminoterminus and the last named amino acid is the carboxyl terminus. Forconvenience, all forms will be collectively referred to hereininterchangeably as "the dipeptide," "EW dipeptide," "R'-Glu-Trp-R," ofwhich L-Glu-L-Trp is a representative and most preferred dipeptide.

Definitions

The following terms are intended to have meanings as follows: namely,

"Polypeptide" is intended to mean a serial array of amino acids of morethan 16 and up to many hundreds of amino acids in length, e.g., aprotein.

"CD2" is intended to mean the lymphocyte cell surface accessory moleculethat is the homo- and heterotypic receptor that mediates binding ofheterologous erythrocytes (e.g., rabbit or sheep erythrocytes) tolymphocytes to form E-rosettes (as disclosed in Paul, W. E. Ed."Fundamental Immunology, 3rd. Edition, Raven Press, N.Y. at page 562;incorporated herein by reference). Lymphocytes having cell surface CD2,when capable of forming rosettes with rabbit erythrocytes are referredto herein as E-rosette forming cells, abbreviated E-RFC.

"CD4⁺ lymphocyte" is intended to mean a lymphocyte having a plurality ofcell surface CD4 molecules as evidenced by binding of an antibodyspecific for CD4 to the cell surface, e.g., monoclonal antibody OKT4(Ortho Diagnostics, Piscataway, N.J.).

"CD8⁺ lymphocyte" is intended to mean a lymphocyte having a plurality ofcell surface CD8 molecules as evidenced by binding of an antibodyspecific for CD8 to the cell surface, e.g., monoclonal antibody OKT8(Ortho Diagnostics, Piscataway, N.J.).

"Reticuloendothelial system" is intended to mean immune system tissuescontaining macrophages, lymphocytes, reticular cells, mast cells,basophils, eosinophils, neutrophils, and the like. Representativecomponents of the reticuloendothelial system include lymph nodes,spleen, thymus, bone marrow, lung, liver, epithelial tissues, lymphaticvessels, blood, and the like.

"Peripheral blood leukocytes", abbreviated PBL, are intended to mean thecellular components of the immune system in blood, e.g., lymphocytes,monocytes, eosinophils, basophils, neutrophils, plasma cells, mast cellprecursors, and the like.

"Immune cell" is intended to encompass the cell types of thereticuloendothelial system, e.g., lymphocytes, mononuclear phagocytes,neutrophils, basophils, eosinophils, mast cells, plasma cells, reticularcells in the spleen, Langerhans cells and δγ-T cell receptor-bearinglymphocytes in the epithelia, Kupfer cells in the liver, and the like.

"Lymphocytes" are intended to encompass T-lymphocytes, (also referred toas T-cells), B-lymphocytes (also referred to as B-cells), naturalkiller-lymphocytes (NK-cells), cytotoxic T lymphocytes (CTL), T-helperlymphocytes, δγ-T-cell receptor bearing cells as well as precursors andactivated derivatives thereof

"Activated lymphocyte" is intended to mean that subset of lymphocyteswhich has been i) exposed to a stimulus, and ii) has been triggered tochange from a metabolically-quiescent cell into a cell with increasedprotein synthesis and/or DNA and RNA synthesis and possibly celldivision. Illustrative stimuli and triggering pathways leading toactivated lymphocytes include interaction of T cell receptors withantigens, interaction of interleukin receptors with interleukins,interaction of growth factor receptors with growth factors, interactionof lymphocyte cell surface determinants with antibody (e.g., anti-CD2)or mitogens (e.g., PHA), and the like.

"Mononuclear phagocytes" are intended to mean cells of themonocyte/macrophage lineage including e.g., monocytes, macrophages,dendritic cells, reticular cells, Langerhans cells, and the like.

"Activated macrophage" is intended to mean a mononuclear phagocytehaving an increased capacity for phagocytosis and intracellular killingof a microbe. Activated macrophages may be recognized, for example, bytesting for phagocytic activity, biochemical markers (e.g.,5'-nucleotidase), or increased oxidative metabolism (e.g., increasedability to reduce nitrotetrazolium blue dye, NBT), and the like.

"Interleukin" is intended to mean an agent released by a first immunecell that affects a biological activity in a second immune cell.Representative interleukins include cytokines such as tumor necrosisfactors (e.g., TNFα and TNFβ), IL-1, IL-2 (also known as T cell growthfactor), IL-3, IL-4, IL-5, IL-6, IFN-γ and the like.

"Growth factor" is intended to mean an agent capable of stimulating anincrease in cell number in a population of immune cells. Representativegrowth factors include granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), granulocyte/macrophagecolony stimulating factor (GM-CSF), stem cell factor (SCF), and thelike. Interleukins that have growth stimulatory activity are included asa subcategory within this usage, (e.g., IL-2, IL-3, 1L-6 and the like),as are compounds having a mitogenic effect on immune cells (e.g., PHA,Con-A, LPS, and the like).

"Anti-microbial cellular and humoral immunity" is intended to mean thatimmunity which ameliorates, makes better, makes normal, or eliminatesone or more clinical or laboratory indicia of disease produced by amicrobe.

"Microbe" is intended to mean an agent that when multiplying in asubject causes a disease. Representative microbes include viruses,bacteria, mycoplasma, mycobacteria, parasites, rickettsia, dengue feveragent, prion disease agent, and the like. Illustrative examples of genusand species of microbes in which the subject methods of the inventionfind uses include: gram positive bacteria (e.g., Staphylococcus,Streptococcus, Actinomyces, and the like), gram negative bacteria (e.g.,Enterobacteriaceae, Bacillus, and the like), acid fast bacteria (e.g.,Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacteriumleprae, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumkansasii, Mycobacterium paratuberculosis), and the like.

Representative examples of clinical indicia of disease include (but arenot limited to) diagnostic manifestations such as redness and swelling,fever, malaise, lethargy, septicemia, pyogenic exudates, and the like.Representative diagnostic manifestations of disease have been classifiedand codified (International Classification of Diseases, ICD-9-CM,Washington, D.C. 1989).

Representative laboratory indicia of disease include (but are notlimited to) "abnormal values" in measurements of i) neutrophil orlymphocyte counts in peripheral blood, ii) hematocrit, iii) serum alphaglobulins or immunoglobulins, iv) expression of one or more cell surfaceaccessory molecules on an immune cell, for example, molecules indicatinga maturation state or activation state of either a T-lymphocyte (e.g.,CD2, CD3, CD4, CD28, and the like) or a B-lymphocyte (e.g., B220,surface Ig, expression of rearrangement-recombinase activating genes inRag 1 and Rag 2, and the like) or a mononuclear phagocyte (e.g.,Ia/Mac-1 expression, 5'-nucleotidase activity and the like), v)synthesis of one or more interleukins (e.g., IL-2, IL-1, TNF, TGF-β,IFN-γ, and the like), vi) synthesis of antibody specific for the subjectmicrobe (e.g., using a diagnostic immunoassay format), vii) phagocyticactivity in an in vitro assay with mononuclear phagocytes or neutrophilsfrom the subject, and viii) splenic mass (e.g., as determined by CATscan or sonography).

"Abnormal" as used herein refers to immune parameters of values that areoutside of the range of values recorded in healthy individuals.

"Normalized" as used herein refers to changes in laboratory or clinicalvalues that are, following treatment, returned to within the normalrange of values recorded for normal healthy subjects.

"Cellular immunity" to a subject microbe is intended to mean thatimmunity which is conferred upon a host by immune cells and which, in anexperimental animal model system, may be transferred from one animal toanother using immune cells that are free of serum, and are free ofB-lymphocytes and/or plasma cells producing an immunoglobulinspecifically reactive with the subject microbe.

"Humoral immunity" to a subject microbe is intended to mean thatimmunity which is conferred upon a host by antibody and which, in anexperimental animal model system, may be transferred from one animal toanother using immune serum that contains an antibody preparation that isfree of cells, or alternatively, may be transferred by B-lymphocytesand/or plasma cells that are producing antibody specifically reactivewith the subject microbe.

"Innate resistance to infection" is used to mean that immunity which ispreexistent in a host before exposure to a microbe such as that which isconferred by Langerhans cells and T-lymphocytes bearing γδ⁺ -T cellreceptor molecules in epithelial tissues, or immune cells in biologicalfluids. In the context of the present disclosure the term is intended toencompass anti-microbial activities of neutrophils, monocytes,platelets, macrophages, dendritic cells, and Langerhans cells, and notthe activities of serum proteins such as coagulation factors,complement, lysozyme and the like.

As used herein, the term "compromised host" is used interchangeably with"compromised subject" to refer to a patient who is at an increased riskof infectious complications because of a deficiency in any of his (orher) defense mechanisms, (e.g., skin barrier, mucous membrane barrier,innate immunity, humoral immunity, or cellular immunity). When her (orhis) defect specifically involves elements of the immune response, theyare termed herein "immunocompromised hosts" or "immunocompromisedsubjects." A subject without an immune defect and without a knowndeficiency in any defense mechanism is referred to hereininterchangeably as "a normal healthy subject," an "immunocompetenthost," or an "immunocompetent subject."

As used herein, the terms "modulator" and "modulating" mean the agentand process of altering a normal or compromised subject's resistance toinfection, e.g., by increasing the flow of mucus, promoting woundhealing of epithelial barriers and the like.

As used herein the terms "immunomodulator" and "immunomodulating" meanthe agent and process of altering a normal or immunocompromisedsubject's immune system, as evidenced by a change in one or morelaboratory measurements from a pre-treatment value to a post-treatmentvalue and/or a change in the subject's ability to combat an infectiousdisease, and to heal tissue. Hence, immunomodulation as used hereinmeans altering one or more cellular and/or humoral immune indices(clinical or laboratory) from a baseline pretreatment value to adifferent value. In one embodiment immunomodulation results instimulation of cellular and/or humoral immunity in an immunodeficientsubject, e.g., a subject exposed to accidental radiation, or treatedwith chemotherapy, radiation therapy, or thymectomy. In anotherembodiment immunomodulation results in depression of cellular and/orhumoral immunity in a subject with a regulatory abnormality in theimmune system, e.g., a subject with an autoimmune disease. The presentinvention encompasses both therapeutic methods of treating theimmunodeficient, immunodepressed, or autoimmune states per se, as wellas prophylactic therapies designed to stimulate cellular and/or humoralimmunity to infectious agents and disease, as well as a treatment ofinfections, diseases or wounds, indirectly by enhancing the immunesystem. This includes enhancing or restoring the subject's immunesystem, as evidenced by measurable blood parameters and/or the patient'simproved ability to combat infection or disease, and the ability to healtissue. The infections may be caused by a variety of microbes, e.g.,viruses, bacteria, mycobacteria, fungus, parasites and the like. Hence,immunomodulation encompasses improvement of the immune system due to animmunodeficient state (e.g., caused by removal of the thymus, exposureto radiation, or acquired during infection with a microbe). Furthermore,the present invention provides methods for modulating the immune systemby lowering blood parameters and other indicia of an autoimmune state ifthese indicia are abnormally elevated. The present invention encompassesboth prophylactic and therapeutic regimens.

"Subject in need thereof" is intended to mean a mammal having one ormore clinical or laboratory indicia of a disease. The subject mayexhibit clinical disease activity or may have a subclinical or latentinfection. Subjects in need thereof include human and non-humanprimates, mammals, domestic animals, livestock, and the like, e.g.,dogs, cats, rodents, birds, horses, cows, pigs and fish.

"R'-Glu-Trp-R"" treatment" is intended to mean a method of delivering toa subject in need thereof a pharmaceutical preparation of R'-Glu-Trp-R",(or derivatives, multimers, analogs, cyclic forms and the like ofFormulas I-III), with the aim of ameliorating, making better/makingnormal, one or more clinical or laboratory indicia of disease in thesubject. The subject methods include delivering the preparation to apatient i) before the disease has been diagnosed, e.g., prophylacticprotocols delivered with the aim of preventing development of thedisease, as well as, ii) after the disease has been diagnosed, e.g.,therapeutic protocols. That the subject treatments have fulfilled theintended aim of treating or preventing the microbial infection in thesubject will be evident by a change (increase or decrease) or completeelimination of one or more clinical or laboratory indicia of disease asset forth above. Representative illness, diseases, and conditions havebeen classified and codified ("International Classification of Diseases;ICD-9-CM, Washington D.C., 1989). The methods of the invention find usein treatment of a variety of disease conditions where it is advantageousto stimulate an immune response. For example, embodiments of theinvention find use in treating infections, e.g., for stimulatingimmunity against the microbe. Representative infections treatable by themethods of the invention include those caused by the following microbes:namely, Mycobacteria genus, gram negative bacteria (e.g., E coli), grampositive bacteria (e.g., Staphylococci), Pseudomonas genus, Hemophilusgenus, Mycoplasma genus, Pneumocystis genus, influenza, rhinovirus, HIV,and the like. Furthermore, the subject compositions may be used to aidhealing of burns, wounds, open sores, sun exposure, local trauma,eczema, psoriasis, and the like. Other illustrative uses include healingbone fractures, lesions, periodontal and gingival diseases, obstetricand gynecologic diseases of the uterus and pregnancy, lymphaticinfections and the like.

Increased Immunity

In one preferred embodiment an R'-Glu-Trp-R" pharmaceutical preparationis administered to an immunocompromised patient in an amount and for atime sufficient to increase, one or more indicia of either cell mediatedimmunity, humoral immunity, or innate resistance to infection, therebyeffecting improvement in the clinical condition of the patient sotreated. In an alternative preferred embodiment, an R'-Glu-Trp-R"pharmaceutical preparation is administered to a patient having anautoimmune disease in an amount and for a time sufficient to decrease,one or more indicia of either cell mediated immunity, humoral immunity,or innate resistance to infection, thereby effecting improvement in theclinical condition of the patient so treated.

Cell-mediated Immunity

The subject indicia of cell mediated immunity preferably include one ormore measures of the number or percentage of immune cells in circulatingperipheral blood of the subject, i.e., leukocytes, lymphocytes,monocytes, T-lymphocytes, B-lymphocytes, stem cells, CD2⁺ -ymphocytes,CD4⁺ -ymphocytes, CD8⁺ -lymphocytes, CD19⁺ lymphocytes, plasma cells,neutrophils, stab neutrophils, segmented neutrophils, basophils,eosinophils, platelets, erythrocytes, and the like.

In the illustrative Examples, the R'-Glu-Trp-R" pharmaceuticalpreparations were administered in clinical tests according to themethods of the invention in an amount and for a time sufficient toincrease the number of: human peripheral blood leukocytes by about1.1-fold to about 1.6-fold (e.g., EXAMPLES 4, 6, and 19), lymphocytes ofby about 1.1-fold to about 2.4-fold (e.g., EXAMPLES 3, 4, 6-7, 16, and19), CD2⁺ -ymphocytes by about 1.1-fold to about 2.6-fold (e.g., EXAMPLE2), CD4⁺ -ymphocytes by about 1.1-fold to about 6.4-fold (e.g., EXAMPLES2-4 and 16), CD8⁺ -lymphocytes by about 1.1-fold to about 2.5-fold(e.g., EXAMPLES 3, 6, and 16), surface immunoglobulin positiveB-lymphocytes by about 1.1-fold to about 1.6-fold (e.g., EXAMPLES 2-4,6, 7, 16, 17 and 19), E-rosette forming T-lymphocytes by about 1.1-foldto about 1.8-fold (e.g., EXAMPLES 4, 6, 9, 16, 19, 23 and 25), and thenumber of neutrophils by about 1.1-fold to about 1.4-fold (e.g.,EXAMPLES 4 and 6).

In other illustrative Examples, the R'-Glu-Trp-R" pharmaceuticalpreparations were administered in clinical tests according to themethods of the invention in an amount and for a time sufficient toincrease the ratio of the percentages of CD4⁺ lymphocytes/CD8⁺lymphocytes by about 1.1-fold to about 1.8-fold (e.g., EXAMPLES 2-4 and25). In still other illustrative Examples, the R'-Glu-Trp-R"pharmaceutical preparations were administered in clinical testsaccording to the methods of the invention in an amount and for a timesufficient to increase the number of monocytes by about 1.1-fold toabout 1.4-fold (e.g., EXAMPLES 4 and 6).

In alternative embodiments the subject indicia of cell mediated immunitypreferably included one or more measures of one or more activities ofimmune cells in circulation in the peripheral blood of the subject,e.g., an activity assay of cytokine (a.k.a. interleukin) integrin oradhesin synthesis by lymphocytes (e.g., IL-1, IL-2, IL-3, IL-4, IL-5,TNF-α, TGF-β, IFN-α, IFN-γ, integrin α₃ β₁, α₆ β₄, α₅ β₁ and the like),a lymphocyte blast transformation assay, an assay for a maturation oractivation state or functional commitment of a T-lymphocyte (e.g., CD2,CD3, CD4, CD8, and the like) or monocyte (e.g., Ia/Mac-1), a monocytephagocytosis assay (e.g., yeast or latex), a marker enzyme assay for anactivation state of a macrophage or a neutrophil (e.g., 5'-nucleotidaseenzyme, nonspecific acid esterase, nitrotetrazolium blue reduction andthe like), an assay for a synthetic activity of the blood cells, anantibody dependent cellular cytotoxicity assay, a natural killer cellassay, a histological assay, an immunohistochemical assay, acytofluorometric assay, and an enzyme-linked immunoassay.

In the illustrative Examples, the R'-Glu-Trp-R" pharmaceuticalpreparations were administered in clinical tests according to themethods of the invention in an amount and for a time sufficient to: (i)increase the percentage of blast transformed lymphocytes after additionof PHA mitogen or a Concanavalin-A mitogen by about 1.1-fold to about1.9-fold (e.g., EXAMPLES 3, 6 and 18); (ii) increase the phagocyticindex (i.e., ingested bacteria, yeast, or latex particles per phagocyticcell) by about 1.1-fold to about 1.8-fold (e.g., EXAMPLES 3 and 6); and(iii) increase NK activity on YAC-target cells of about 1.1-fold toabout 1.5-fold (e.g., EXAMPLE 6).

Yet a third alternative embodiment of the subject indicia of cellmediated immunity preferably includes one or more measures of delayedtype hypersensitivity, e.g., skin test diameter in response to achallenge with a bacterial, parasitic, or cellular antigen as determinedby measuring induration at 36 hours after an intradermal injection ofantigen. In the illustrative Examples, the R'-Glu-Trp-R" pharmaceuticalpreparations were administered in clinical tests according to themethods of the invention in an amount and for a time sufficient toincrease the delayed type hypersensitivity skin test diameters insubjects so treated by about 1.1-fold to about 3.3-fold.

Humoral Immunity

The subject indicia of humoral immunity preferably includes one or moremeasures of either the concentration of immunoglobulin IgG, IgA, IgM,IgD in a sample of a biological fluid (e.g., blood, plasma, saliva, andthe like), or alternatively, a number or percentage of CD4⁺ T-helperlymphocytes, plasma cells, or B-lymphocytes in a sample of peripheralblood. The subject indicia of humoral immunity may most preferablyinclude one or more measures of either the concentration of an antigenspecific immunoglobulin that is capable of binding an antigen in animmunoassay.

In alternative embodiments the subject indicia of humoral immunitypreferably include one or more measures of one or more activities ofimmune cells in circulation in the peripheral blood of the subject,e.g., an assay of immunoglobulin synthesis, an assay for a maturation oran activation state in a B-lymphocyte (e.g., B220, surface Ig, Rag 1,Rag 2, and the like), monocyte (e.g., Ia/Mac-1), or T-helper lymphocyte(e.g., CD4). An alternative embodiment of the subject indicia of humoralimmunity preferably includes one or more measures of immediate or Arthustype hypersensitivity, e.g., skin test diameter in response to achallenge with a bacterial, parasitic, or cellular antigen as determinedby measuring erythema at 3-20 hours after subcutaneous injection ofantigen).

Innate Resistance to Infection

The subject indicia of innate resistance to infection preferablyincludes one or more measures of the concentration of a blood protein ina sample of biological fluid, i.e., an immunoglobulin (e.g., IgA orIgM), a lysozyme (e.g., salivary lysozyme), a cytokine (e.g., IL-2,IL-1, TNF and the like), an interferon (e.g., IFN-α₁, IFN-α₂, IFN-β,IFN-γ, and the like), a complement protein (e.g., C3, C3b, C3a, C5a,Factor B, and the like), a coagulation protein (e.g., fibrinogen, fibrindegradation products, von Willebrand's Factor (vWF), tissue factor,thrombospondin, platelet factor 4, and the like), a fibrinolytic systemprotein (e.g., plasminogen, tissue plasminogen activator, and the like),an enzyme inhibitor (e.g., antithrombin III, α₁ -antitrypsin, α₂-macroglobulin, C3b-inactivator and the like), a bradykinin systemprotein, a hormone (e.g., insulin, a steroid hormone, and the like), anda receptor protein (e.g., a soluble CD4, or IL-1 or IL-2 receptor, andthe like).

In the illustrative Examples, the R'-Glu-Trp-R" pharmaceuticalpreparations were administered in clinical tests according to themethods of the invention in an amount and for a time sufficient todecrease coagulation and/or to increase fibrinolysis. The decrease incoagulation was assayed in illustrative Examples by measuring: (i) thelevels of coagulation enzyme inhibitors in peripheral blood, i.e.,antithrombin III, (abbreviated ATIII), α₁ -antitrypsin, and α₂-macroglobulin; (ii) the levels of fibrin degradation fragments inblood; (iii) the clotting activities of blood, i.e., prothrombin time,(PT), PTT, citrated blood clotting time, and the like; and, (iv) thefibrinolytic activity of blood.

In alternative embodiments, the subject indicia of innate resistance toinfection preferably include one or more measures of one or moreactivities of immune cells in circulation in the peripheral blood of thesubject, e.g., an assay for lymphocytes (e.g., CD28), a maturation or anactivation state in a B-lymphocyte (e.g., B220, surface Ig, Rag 1, Rag2, and the like), monocyte (e.g., Ia/Mac-1), or T-helper or T-suppressorlymphocytes (e.g., CD4 and CD8, respectively), or complement receptors(e.g., CR1, CR2, CR3, and the like).

Treatment by L-Glu-L-Trp

The subject immunocompromised patients amenable to treatment by one ormore of the embodiments of the invention include but are not limited to:(i) patients having a bacterial infection, viral infection, mycoplasmainfection, parasitic infection, opportunistic infection, pneumocystisinfection, cytomegalovirus infection, herpes virus infection,mycobacterium infection, or human immunodeficiency virus infection; (ii)patients exposed to radiation or one or more chemotherapeuticantiproliferative drugs; (iii) patients having a transplant, cancer orautoimmune disease, (i.e., see Frank, et al., eds., SAMPTER'SIMMUNOLOGICAL DISEASES, Little, Brown N.Y. 1994; sytemic lupuserythematosus, rheumatoid arthritis, Sjogren's syndrome, psoriasis,epidermolysis bullosa, or type 1 insulin-dependent diabetes mellitus,(IDM); (iv) patients having a primary or a secondary immune deficiencydisease, (i.e., see Frank, et al., eds., SAMPTER'S IMMUNOLOGICALDISEASES, Little, Brown, N.Y. 1994; (v) patients having a staphylococcalinfection (e.g., pyoderma, furunculitis, cellulitis, eczema, acnevulgaris); (v) patients having gingivitis, dental caries, or periapicalgranulomas, (vi) patients having a gynecological infection, pelvicinflammatory disease (e.g., cervicitis, vaginitis, tubular or ovarianabscess or an adnexal abscess; (vii) patients having having-lymphangitisor an infralymphatic infection, (viii) patients having an acute orchronic respiratory disease, upper airways disease, e.g., sinusitis orperisinusitis, rhinovirus or influenza infection, pleuritis, and thelike); (ix) patients having chronic eye-ear-nose or throat infections(e.g., otitis media, conjunctivitis, uveitis or keratitis); (x) patientshaving bronchial allergy and/or asthma; (xi) patients having a chronicliver infection (e.g., chronic hepatitis); and (xii) patients who are atan increased relative risk of developing an infection or an autoimmunedisease (e.g., relative of of patients with IDDM who are at an increasedrelative risk of developing the disease).

In one of the two alternative preferred embodiments of the invention,(above) an R'-Glu-Trp-R" pharmaceutical preparation is administered to apatient having an autoimmune disease in an amount and for a timesufficient to decrease, one or more indicia of either cell mediatedautoimmunity (e.g., autoimmune T-lymphocytes), humoral autoimmunity(e.g., IgG or IgM rheumatoid factor, IgG or IgM binding nucleic acidantigens, and anti-erythrocyte antibodies), or nonspecific inflammation(e.g., complement or neutrophil mediated tissue destruction), therebyeffecting improvement in the clinical condition of the patient sotreated.

In the illustrative Examples, below, the R'-Glu-Trp-R" pharmaceuticalpreparations were administered in clinical tests according to themethods of the invention in an amount and for a time sufficient to: (i)decrease elevated polymorphonuclear leukocytes counts in peripheralblood by about 1.1-fold to about 1.7-fold; (ii) decrease elevatedmononuclear phagocyte counts in peripheral blood by about 1.1-fold toabout 1.3-fold; (iii) decrease elevated B-lymphocytes in peripheralblood about 1.1-fold to about 1.9-fold; (iv) decrease elevated CD4⁺T-lymphocyte counts in peripheral blood by about 1.1-fold to about1.3-fold; and (v) decrease spontaneously elevated blast transformationof lymphocytes induced by PHA or Concanavalin A of by about 1.1-fold toabout 1.7-fold.

In still another preferred embodiment of the invention, an R'-Glu-Trp-R"pharmaceutical preparation is administered to a patient having asystemic toxicity (e.g., febrile, jaundiced, and the like), therebyeffecting improvement in the clinical condition of the patient sotreated. The R'-Glu-Trp-R" pharmaceutical preparation is administered ata dose and for a time sufficient to decrease the blood level of an acutephase protein in a sample of a biological fluid collected from thesubject. Representative examples of acute phase proteins includeprealbumin, orosomucoid, alpha₁ -antitrypsin, alpha2-macroglobulin,ceruloplasmin, complement C3, and transferrin.

In a preferred embodiment, a treatment regimen consists of administeringa dose of about 10 μg per 1 killogram body weight to about 1 mg per 1 kgbody weight daily over a period of 1 day to about 30 days to thesubject. In preferred embodiments, the subject dose is administeredeither as a single daily intramuscular dose of the R'-Glu-Trp-R"pharmaceutical preparation, or as a single daily intranasal dose of theR'-Glu-Trp-R" pharmaceutical preparation. The subject dose is preferablyformulated as an injectable, inhalant, nose drop, or mucosal spraysolution containing about 0.001% to about 0.01% of the R'-Glu-Trp-R"pharmaceutical preparation. Alternatively, the formulation of theR'-Glu-Trp-R" pharmaceutical preparation may preferably be incorporatedinto a unit dose delivery form, e.g., a tablet, a suppository, acapsule, an eye film, or into a paste or ointment, e.g., a toothpaste, adermal ointment, or water-soluble cream base. A most preferred unit doseform is for delivery of about 0.01 mg of the R'-Glu-Trp-R"pharmaceutical preparation.

In one preferred embodiment, an R'-Glu-Trp-R" pharmaceutical preparationis administered to an immunocompromised patient in an amount and for atime sufficient to increase one or more indicia of either cell mediatedimmunity, humoral immunity, or innate resistance to infection. Thesubject treatment of an immunocompromised patient is effective toimprove the clinical condition of the patient so treated. In analternative preferred embodiment, an R'-Glu-Trp-R" pharmaceuticalpreparation is administered to a patient having an autoimmune disease inan amount and for a time sufficient to decrease one or more indicia ofeither cell mediated immunity, humoral immunity, or innate resistance toinfection. The subject indicia of cell mediated immunity, humoralimmunity, or innate resistance to infection is determined by obtaining asample of blood or tissue from the patient so treated and determining anumber (or percentage of immune cells) or determining a functionalactivity of the immune cells in an in vitro assay. The frequency of thetreatment and/or dosage of the R'-Glu-Trp-R" may be adjusted by thetreating physician until the subject indicia of immunity is within adesired range. Treatment of an immunocompromised patient or patient withan autoimmune disease according to the method of the invention iseffective to improve the clinical condition of the patient.

Assays to Determine Modulation of Immunity

"Indicia of cell mediated immunity" is used herein to mean one or moremeasures ofa number or percentage of immune cells in circulation in theperipheral blood of a subject treated according to the methods of theinvention, e.g., a number or percentage of leukocytes, lymphocytes,monocytes, T-lymphocytes, B-lymphocytes, stem cells, CD2⁺ -lymphocytes,CD4⁺ -lymphocytes, CD8⁺ -lymphocytes, CD19⁺ -lymphocytes, plasma cells,neutrophils, stab neutrophils, segmented neutrophils and the like.

Illustrative uses of the methods of the invention are disclosed in theExamples section, where an R'-Glu-Trp-R" pharmaceutical preparation wasadministered according to the methods of the invention in an amount andfor a time sufficient to increase the number of: peripheral bloodleukocytes by about 1.1-fold to about 1.4-fold (e.g. see EXAMPLES 6 and19); lymphocytes by about 1.1-fold to about 1.7-fold (e.g. see EXAMPLES6, 7, 16); CD2⁺ -lymphocytes by about 1.1-fold to about 2.2-fold (e.g.see EXAMPLE 2); CD4⁺ -lymphocytes by about 1.1-fold to about 3.2-fold(e.g., see EXAMPLES 3, 4, and 16); CD8⁺ -lymphocytes by about 1.1-foldto about 1.8-fold (e.g., see EXAMPLES 3 and 6); cell-surface Ig⁺B-lymphocytes by about 1.1-fold to about 1.6-fold (e.g., see EXAMPLES 6,7, 16, and 19); E-rosette forming T-lymphocytes by about 1.1-fold toabout 1.8-fold (e.g., see EXAMPLES 4, 6, 7, 9 and 19); polymorphonuclearneutrophils by about 1.1-fold to about 1.3-fold (e.g., see EXAMPLES 4and 6); and/or monocytes by about 1.1-fold to about 1.4-fold (e.g., seeEXAMPLES 4 and 6).

In other Examples, an R'-Glu-Trp-R" pharmaceutical preparation wasadministered in an amount and for a time sufficient to increase theratio of the numbers of CD4⁺ -lymphocytes/ CD8⁺ -lymphocytes by about20% to about 40% from the pretreatment value (e.g., see EXAMPLES 2, 3and 4; % increase=(ratio after treatment)-(ratio beforetreatment)/(ratio before treatment)). In still other Examples, anR'-Glu-Trp-R" pharmaceutical preparation was administered in an amountand for a time sufficient increase, (or in certain patient populations,to decrease), one or more measures of functional activity of immunecells selected from among: lymphocyte blastogenesis with PHA(phytohaemagglutinin) or Con A (concanavalin A); lymphocyte cytotoxicactivity (e.g., CTL, ADCC, antibody dependent cytotoxicity, and NK cellcytotoxicity assay); monocyte phagocytic activity; differentiated oractivated mononuclear phagocytes in tissues or peripheral blood (i.e.,identified using markers such as nonspecific acid esterase,5'-nucleotidase, nitrotetrazolium blue reduction and the like); and,neutrophil phagocytic activity. In still other Examples, anR'-Glu-Trp-R" pharmaceutical preparation was administered in an amountand for a time sufficient to alleviate infection and decrease the levelof potentially inflammatory cytokines (interleukins) produced bylymphocytes and monocytes (e.g., LMIR; EXAMPLES 3, 9, and 16).Monitoring efficacy of an R'-Glu-Trp-R" treatment by measuring increasedor decreased interleukin production may vary dependent upon theparticular disease state, patient condition, and cytokine measurement:e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, TNFα, TGFβ, IFNα, IFNγ.

Other assays useful for monitoring efficacy of an R'-Glu-Trp-R"treatment may include measuring expression of cell-surface integrins onlymphocytes, mononuclear phagocytes, or neutrophils (e.g., α₃ β₄, α₃ β₁,α₅ β₁ and the like). Still other assays useful for monitoring efficacyof an R'-Glu-Trp-R" treatment may include measuring expression of acell-surface marker assay for the maturational, activational orfunctional conmmitment state of a lymphocyte (e.g., CD 2, CD 3, CD 4, CD8, CD 26, CD 45 and the like) or a monocyte (e.g., Ia/Mac-1).

Histological and immunohistochemical assays conducted with biopsysamples, and delayed type hypersensitivity skin test reactions are alsouseful for monitoring efficacy of an R'-Glu-Trp-R" treatment in certainpatients (e.g., EXAMPLES 3 and 18). In the Examples section, anR'-Glu-Trp-R" pharmaceutical preparation was administered in clinicaltests according to the methods of the invention in an amount and for atime sufficient to: (i) increase the percentage of blast transformedlymphocytes after addition of PHA or Con-A mitogen by about 1.1-fold toabout 1.9-fold (e.g., see EXAMPLES 3, 6, and 18); (ii) increase thephagocytic index of mononuclear phagocytes by about 1.1-fold to about1.8-fold (e.g., see EXAMPLES 3 and 6); (iii) increase NK activity onYAC-target cells by about 1.1-fold to about 1.5-fold (e.g., see EXAMPLES1 and 6 ); (iv) decrease endogenous cytokine production in an assay forleukocyte migration inhibition factor (LMIR) by about 40% to about 75%(e.g., see EXAMPLES 3, 9 and 16); and, (v) increase a delayed typehypersensitivity skin test diameter by about 1.1-fold to about 3.3-fold(e.g., see EXAMPLES 3 and 18).

"Indicia of humoral immunity" is used herein to mean one or moremeasures of a number or percentage of lymphocytes or plasma cells,and/or one or more immunoglobulin levels in a sample of peripheral bloodfrom a subject treated according to the methods of the invention. In thelatter case, the number or percentage of B-lymphocytes, plasma cells,surface Ig⁺ -lymphocytes, Fc-receptor lymphocytes, CD19⁺ -lymphocytes,B220⁺ -lymphocytes, Rag-1⁺ - or Rag-2⁺ -lymphocytes and the like may bedetermined; and/or, the levels of IgA, IgD, IgG, IgM or IgE.

"Indicia of innate resistance to infection" is used herein to mean oneor more measures of a number or percentage of immune cells or theirbiosynthetic products in a sample of tissue or blood in a tissue orblood sample of a subject treated according to the methods of theinvention. In the latter case, "immune cells" include (but are notlimited to) δγ⁺ -T-lymphocytes, neutrophils, mononuclear phagocytes,Mac-1⁺ -macrophages, reticular cells, dendritic cells, and the like;and, "biosynthetic products of immune cells" include (but are notlimited to) immunoglobulins (e.g., IgA, IgD, IgE, IgK IgG and the like),lysozymes (e.g., salivary lysozyme), interferons (e.g., IFN-α₁, IFN-α₂,IFN-β, IFN-γ and the like), complement proteins (e.g., C3, C3b, C3a,C5a, Factor Bb and the like), coagulation proteins (e.g., fibrinogen,fibrin degradation products, vWF, tissue factor, thrombospondin,platelet factor 4, and the like), fibrinolytic proteins (e.g.,plasminogen, tissue plasminogen activator, and the like), enzymeinhibitors (e.g., antithrombin III, α₁ -antitrypsin, α₂ -macroglobulin,C3b-inactivator and the like), arachidonic acid metabolites (e.g.,prostaglandins, and the like), bradykinin system proteins, and receptors(e.g., soluble CD 4, IL-1 receptor, IL-2 receptor and the like).

Methods of Treatment

Embodiments of the invention provide methods useful for treating asubject to induce a heightened state of anti-microbial cellular orhumoral immunity by administering a pharmaceutical preparation ofR'-Glu-Trp-R". Pharmaceutical preparations containing both R'-Glu-Trp-R"and a second agent are also envisaged, including a variety of differentmixtures. The "subject in need of" the treatments of the invention arepreferably man and domestic animals having one or more microbialinfections that include, but are not limited to, subjects infected withthe following infectious agents: bacteria, viruses, mycobacteria,parasites, opportunistic bacteria or viruses (e.g., pneumocystis,cytomegalovirus, herpes virus, mycoplasma, and the like). Representativeexamples of "subjects in need thereof" include the following: (i)individuals infected with human immunodeficiency virus and having asecondary opportunistic infection; (ii) individuals exposed to radiationand/or one or more chemotherapeutic agents (e.g., cancer patients);(iii) individuals undergoing immunosuppressive drug therapy (e.g.,following transplantation or for treatment of an autoimmune disease suchas rheumatoid arthritis, systemic lupus erythematosus, type 1 insulindependent diabetes (IDDM), Sjogren's syndrome, and the like); (iv)individuals having a primary or secondary immune deficiency disease; (v)individuals having a staphylococcal infection (e.g., pyoderma,firunculitis cellulitis, eczema, acne vulgaris) or psoriasis; (vi)individuals with gingivitis, dental caries, or periapical granulomas;(vii) individuals with lymphangitis or infralymphatic infection; (viii)individuals with acute or chronic respiratory disease and/or upperairways disease (e.g., sinusitis or perisinusitis, rhinovirus orinfluenza infection, bronchitis, pharyngitis, and the like), (ix) lowerairways disease (e.g., pneumonia or pleuritis); (x) individuals withacute or chronic eye, nose or throat infections (e.g., otitis media,conjunctivitis, uveitis, or keratitis); (xi) individuals with bronchialallergy and/or asthma; (xii) patients having a chronic liver infection(e.g., hepatitis); and, (xiii) individuals at an increased relative riskof developing an infection or an autoimmune disease (e.g., relatives ofa Type 1 IDDM patient).

"Immune deviation" is used herein to mean that the treatment method ofthe invention may be effective to change the effector elements involvedin an ongoing immune response but the term as used herein does not meaneffecting a quantitative increase or decrease in an ongoing immuneresponse. Representative examples of immune deviation include affectingin the subject so treated (i) a change from an ongoing humoral immuneresponse to a cell-mediated immune response, (ii) a change from anIgE-mediated immune response to an IgG-mediated immune response; (iii) achange from a first microbial antigen to a second microbial antigen;(iv) a change from a cytotoxic lymphocyte response to a macrophagemediated immune response; and, (v) a change from an autoimmune cytotoxiclymphocyte response to production of an IgG that blocks cytotoxickilling of tissue cells. The treatment methods of the invention mayeffect immune deviation in a treated subject. Immune deviation in thetreated subject may be determined by evaluating cell mediated andhumoral immunity, prior to and after treatment.

As disclosed above, the "subject in need thereof" may be animmunocompromised subject such an individual have an underlyingimmunodeficiency syndrome occasioning frequent, recurrent, orintermittent microbial infections. Underlying patient syndromes include(but are not limited to) hereditary and acquired immunodeficiencysyndromes such as primary immunodeficiency syndromes (e.g., DiGeorgeSyndrome, severe combined immunodeficiency syndrome, combinedimmunodeficiency, X-linked lymphoproliferative disease); syndromesresulting from an underlying autoimmune disease (e.g., diabetes,systemic lupus erythematosus, Sjogren's syndrome); syndromes resultingfrom an underlying infection (e.g., HIV infection); syndromes resultingfrom a toxic reaction to a drug or a chemical; or secondaryimmunodeficiency disorders such as those disclosed in EXAMPLES 2-3,12-13, 24, and 28.

The subject treatment methods of the invention preferably stimulateincreased cellular, humoral or innate immunity, and thereby ameliorateor eliminate one or more "clinical or laboratory indicia of disease,"e.g., redness and swelling, fever, malaise, septicemia, pyogenicexudates, and the like. The subject treatment methods of the inventionmay also restore one or more measurements of a "laboratory indicia ofdisease," i.e., changing a measurement value from an abnormal value to amore normal value; or alternatively, the subject treatments may increasea laboratory value above the normal range, i.e., to achieve an increasedanti-microbial immunity. Representative examples of changes in alaboratory indicia of disease include (but are not limited to) thefollowing: (i) decreasing an elevated neutrophil or lymphocyte count ina sample of peripheral blood into the normal range of values; (ii)increasing an abnormally low hematocrit; (iii) decreasing an elevatedserum alpha globulin level in a serum sample (e.g., an acute phasereactant measurement); (iv) decreasing an elevated IgM immunoglobulinlevel or changing an IgG₁ /IgG₂ ratio in a serum sample; (v) increasingabnormally low expression of one or more cell surface accessorymolecules in a blood sample of immune cells (e.g., CD2, CD3, CD4, CD26,CD28, CD45, B220, surface Ig, Rag 1 and Rag 2, Ia/Mac-1 and the like),(vi) increasing biosynthesis of one or more interleukins (e.g., IL-2,IL-1, TNF, TGF-β, IFN-γ, and the like) in a sample of immune cellsisolated from the subject; (vi) increasing biosynthesis of antibodyspecific for the subject microbe (e.g., using a diagnostic immunoassayformat) in a serum sample; (vii) increasing phagocytic activity in an invitro assay with mononuclear phagocytes or neutrophils from a sample ofblood; (viii) decreasing an abnormally enlarged spleen or increasing asmall spleen to more effectively combat infection (e.g., as determinedby CAT scan or sonography); (ix) increasing the proportion ofFc-receptor-bearing lymphoid cells in spleen, thymus, and/or bone marrowsample; (x) increasing Fc-receptor-bearing lymphoid cells in a sample ofperipheral blood; (xi) increasing T-lymphocytes in a spleen biopsyspecimen; (xii) increasing the number of neutrophils in a measuredvolume of a tissue infiltrate in response to an inflammatory agent;and/or (xiii) increasing the percentage of phagocytically active cellsin a neutrophil infiltrate sample from the subject.

Utility of Treatment

The subject methods of the invention find a variety of prophylactic andtherapeutic uses in treatment of immune pathophysiologic conditions inman and domestic animals. In certain embodiments the methods of theinvention find use during in vitro maintenance and expansion of bonemarrow, peripheral blood leukocytes, CD34⁺ lymphocytes, and other immunecells, such as may occur prior to autologous or allogenic bone marrowtransplantation.

In one preferred embodiment, an R'-Glu-Trp-R" pharmaceutical preparationis administered to an individual with a diagnosed autoimmune disease inan amount and for a time sufficient to decrease one or more laboratoryindicia of the disease state in the patient. Representative examples ofthe laboratory indicia include the following: (i) levels of IgG and/orIgM rheumatoid factor in patients with rheumatoid arthritis (RA); (ii)levels of anti-red cell antibodies in patients with systemic lupuserythematosus (SLE); (iii) levels of complement activation fragments(e.g., C3a, C5a, C3b, SC5b-9, Bb and the like) in patients with IDDM, RAor SLE; (iv) levels of fibrin degradation fragments in patients withjuvenile-onset RA; (v) levels of spontaneous lymphocyte blastogenesis invitro (e.g., ³ H-thymidine incorporation).

Illustrative uses of the subject treatment methods in several differentpatient populations resulted in: (i) a decrease of about 1.1-fold toabout 1.7-fold in peripheral blood polymorphonuclear leukocyte countsfrom an elevated pretreatment value to a lower post-treatment value inpatients with chronic staphylococcal infections and acute osteomyelitis(e.g., see EXAMPLES 6 and 16); (ii) a decrease of about 1.1-fold toabout 1.3-fold in peripheral blood mononuclear phagocyte counts from anelevated pretreatment value to a lower post-treatment value in patientswith chronic staphylococcal infections (e.g., see EXAMPLE 6); (iii) adecrease of about 1.1-fold to about 1.9-fold in B-lymphocytes from anelevated pretreatment value to a lower post-treatment value in patientswith chronic staphylococcal or hepatitis infection (e.g., see EXAMPLES 6and 23); and, (iv) a decrease of about 1.1-fold to about 1.3-fold in CD4⁺ -lymphocytes in peripheral blood from an elevated pretreatment valueto a lower post-treatment value in patients with SLE (e.g., see EXAMPLE6).

In another preferred embodiment, an R'-Glu-Trp-R" pharmaceuticalpreparation is administered to an individual exhibiting systemictoxicity, (e.g., febrile, jaundiced, alternating chills and fever), inan amount and for a time sufficient to decrease one or more laboratoryindicia of the disease state in the patient. Representative examples ofthe laboratory indicia include measurements of fibrin degradationproducts (FDP), antithrombin-III, orosomucoid, prealbumin, alpha₁-antitrypsin, alpha₂ -macroglobulin, ceruloplasmin, complement C3 andtransferrin. In a preferred embodiment a treatment regimen according tothe method of the invention consists of administering to the subject inneed thereof a dose of about 10 μg per 1 ldlogram of body weight toabout 1 mg per 1 kg body weight once daily an each of about 1 day toabout 30 days. In most preferred embodiments the subject dose isadministered either as a single daily intramuscular dose, or as a singledaily intranasal dose, of an R'-Glu-Trp-R" pharmaceutical preparation.In the latter case the subject dosage form is preferably either asterile injectable solution, or inhalant, nose drop or mucosal spraysolution containing about 0.001% weight to about 0.01% of theR'-Glu-Trp-R" pharmaceutical preparation. Alternatively, the formulationof the R'-Glu-Trp-R" pharmaceutical preparation may be in a unit dosedelivery form, e.g., a tablet, capsule, suppository, eye film, paste orointment. A most preferred unit dose form delivers about 0.01 mg of theR'-Glu-Trp-R" pharmaceutical preparation.

It is an object of the present invention to provide methods andR'-Glu-Trp-R" pharmaceutical compositions, (of which the dipeptideL-Glu-L-Trp, its analogs and derivatives, multimers and cyclized formsare representative examples), that have immunomodulatory activity formedical and veterinary uses, e.g., treatment of infections, diseases,wounds, burns, frost bites, and the like. It is also an object of thepresent invention to provide therapeutic methods for treatment ofimmunodepressed and immunodeficient states. It is yet another object ofthe present invention to provide methods for preventing and treatinginfections in a subject including opportunistic infection in animmunodeficient or immunodepressed subject.

Treatment for Immune Disorders

In a representative prophylactic treatment regimen, the subjectcompositions of the invention are administered to a patient susceptibleto, or otherwise at risk, for infection, anemia, or immune regulatorydisorder that may be amenable to treatment by the subject methods of theinvention in an amount defined as a "prophylactically effective dose.""Prophylactically effective dose" is used herein to mean an amountsufficient to protect the subject against development of a disease,wherein the amount will depend on the patient's state of health andweight, but will generally fall within the ranges described herein fortherapeutic use. Prophylactic administration may be particularlydesirable for hosts that have been exposed or at risk for exposure ofinfectious diseases, e.g., health-care workers, travelers, familymembers of infected individuals, immunosuppressed persons, and the like.The compositions of the present invention can be used for prophylaxisagainst agents of common illnesses such as rhinoviruses,orthomyxoviruses, adenoviruses, a-hemolytic Streptococcus,a-Staphylococcus, and the like. The compositions of the presentinvention can be administered for surgical prophylaxis to lessen therisk of infectious complications. The compositions can also be used toinhibit organ rejection. Such organs can include skin, heart, lung,kidney, bone, liver, pancreas, tendon, and the like. The presentcompositions are particularly useful when used prophylactically toinhibit rejection of skin grafts.

In a representative therapeutic treatment regimen, a pharmaceuticalpreparation of the present invention is administered alone, oroptionally with a second pharmaceutical agent, i.e., the latter twoagent therapy is termed herein "combined therapy". In a combined therapythe subject R'-Glu-Trp-R" composition is preferably administered withone or more antibiotics, anti-viral compounds, or anti-fungal compounds,anti-parasitic agents, anti-inflammatory agent, or chemotherapeuticcompounds. The subject compositions may be administered either inconjunction with the second treatment modalities, or separately, e.g.,at different times or in different syringes or tablets. Often, the doseof the additional agents may be less than standard dosages (i.e.,because of the immunostimulatory effects of the subject compositions.Often, R'-Glu-Trp-R" is administered in a combined therapy with ananti-infective agent such as one or more antibiotics (i.e.,anti-inflammatory agents, vaccines, antihistamines, immunomodulators,chemotherapeutic agents and the like. Illustrative combined treatmentswith R'-Glu-Trp-R" may include, e.g., anti-inflammatory agents such assalicylates, Diclofenac sodium, Etodolac, fenoprofen calcium,flurbiprofen, ibuprofen, ketoprofen, meclofenamate sodium monohydrate,nabumetone, naproxen, naproxen sodium, oxaprozin, phenylbutazone,piroxicam, sulindac, tolmetin sodium, hydroxychloroquine sulfate,methotrexate, penicillamine, sulfasalazine, aurothioglucose, gold sodiumthiomalate, auranofin, adrenal corticosteroids, azathioprine,colchicine, corticotropin, allopurinol, probenecid, sulfinpyrazone, andthe like; antihistamines such as amino alkylethers, clemastine fumarate,tripelennamine citrate, tripelennamine hydrochloride, pyrilaminemaleate, chlorpheniramine maleate, brompheniramine maleate,dexchlorpheniramine maleate, triprolidine hydrochloride, methdilazine,methdilazine hydrochloride, promethazine hydrochloride, trimeprazinetartrate, azatadine maleate, cyproheptadine hydrochloride, hydroxyzinehydrochloride, hydroxyzine pamoate, acrivastine, astemizole, cetiriziehydrochloride, levocabastine hydrochloride, loratadine, terfenadine,ethanolamines, ethylenediamine, alkylamines, phenothiazine, and thelike; immunomodulators such as, glucocorticoids, acetate, cypionate,sodium phosphate, sodium succinate, tebutate, azathioprine, azathioprinesodium, chlorambucil, cyclophosphamide, methotrexate, methotrexatesodium, cyclosporine, muromonab-CD3, aldesleuin, BCG vaccine,interferon-γ_(1b), levamisole, bovine pegademase, sargramostin,filgrastim, immune globulin, lymphocyte immune globulin, muramyldipeptide, and thymic hormones.

Illustrative combined treatments with R'-Glu-Trp-R" may includeadministration of a chemotherapeutic agent as the second agent.Representative chemotherapeutic agents so useful include: chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine hydrochloride, melphalan,thiotepa, busulfan, procarbazine hydrochloride, carmustine, lomustine,streptozocin, cisplatin, carboplatin, dacarbazine, altretamine, mesna,methotrexate, leucovorin calcium, cytarabine, floxuridine, fluorouracil,cladribine, fludarabine, mercaptopurine, pentostatin, thioguanine,hydroxyurea, bleomycin sulfate, dactinomycin, daunorubicinhydrochloride, doxorubicin hydrochloride, idarubicin hydrochloride,mitomycin, mitoxantrone hydrochloride, plicamycin, vinblastine sulfate,vincristine sulfate, etoposide, paclitaxel, teniposide, asparaginase,prednisone, prednisolone, dexamethasone, methylprednisolone,diethylstilbestrol, chlorotrianisene, conjugated estrogen, esterifiedestrogens, estone, ethinyl estradiol, estramustine phosphate sodium,tamoxifen citrate, fluoxymesterone, methyltestosterone, testolactone,testosterone propinate, flutamide, goserelin acetate, leuprolideacetate, hydroxyprogesterone caproate, medroxyprogesterone acetate,megestrol acetate, aminoglutethimide, mitotane, aldesleukin,interferon-α_(2a), BCG, isotretinoin, levamisole, octreotide acetate,cyclophosphamide, ifosfamide, mechlorethamine hydrochloride, melphalan,nimustine, semustine, iproplatin, sodium phosphate P³², chromicphosphate P³², floxuridine, azacitidine, tegafur, cladribine,fludarabine phosphate, pentostatin, thioguanine, tiazofurin,hydroxyurea, caracemide, buthionine sulfoximine, eflornithinehydrochloride, mitoguazone, phosphonoacetyl, brequinar sodium,doxorubicin hydrochloride, idarubicin hydrochloride, epirubicinhydrochloride, menogaril, razoxane, dactinomycin, mitomycin, plicamycin,didemtin B, echinomycin, deoxyspergualin, mitoxantrone hydrochloride,amsacrine, amonafide, merbarone, piroxantrone hydrochloride, vindesinesulfate, etoposide, teniposide, homoharringtonine, asparaginase,mitotane, estramustine phosphate sodium, leuprolide acetate, buserelinacetate, aminoglutethimide, interferon Alpha-2b, interferon Beta,interleukin 2, tumor necrosis factor, live BCG, BCG vaccine, monoclonalantibodies, flavone acetic acid, hexamethylene-bis-acetamide,isotretinoin, levamisole hydrochloride, N-methyformamide, octreotideacetate, and the like.

Representative uses of the subject methods of the invention in treatingan immunocompromised subject include treatments designed to ameliorateclinical symptoms resulting from administration of chemotherapeuticcompounds, e.g., during treatment of malignancy, graft-versus-hostdisease, autoimmune states, and the like. Autoimmune states in whichtreatment the methods of the present invention may prove useful includediseases such as rheumatoid arthritis, systemic lupus erythematosus,Reiter's Syndrome, psoriasis, ankylosing spondylitis, Sjogren'ssyndrome, sicca syndrome, mixed connective tissue disorder, multiplesclerosis, diabetes mellitus, and the like (see Frank, et al., eds,SAMTER'S IMMUNOLOGICAL DISEASES, Little, Brown, N.Y. 1994). Theimmunomodulating properties of the pharmaceutical compositions of thepresent invention provide a means for ameliorating, i.e., makingbetter/making normal, one or more symptoms of disease in a subject.

Treatment of Infectious Diseases

A variety of disease states may be treated by the subject methods of theinvention. Infectious diseases may be treated. The infections may bebacterial, viral, fungal, or parasitic. The methods may be practiced inimmunocompromised or immunocompetent hosts. Illustrative uses of themethods of the invention in treating patients with bacterial diseasesare disclosed in the Examples section as follows: in treating patientswith staphylococcal skin diseases (EXAMPLE 6); in treating patients withpelvic inflammatory diseases including complications of pregnancy,post-partem infections, and uterine infections (EXAMPLE 7); in treatingpatients with gastrointestinal diseases including Shigella dysentery(EXAMPLE 23); and, opportunistic infections (EXAMPLE 13).

Localized or disseminated infections may be treated by the presentmethods. The infections may be in any organ, tissue or body cavity,e.g., lungs, bone, kidney, central nervous system, heart, skin and softtissues (e.g., post-traumatic infections), reproductive organs(orchitis, pelvic inflammatory diseases, and the like), liver, and thelike. Illustrative uses of the subject methods in treatment of localizedand disseminated infections are disclosed in the Examples section asfollows: nose, ear and throat infections, sinusitis, pleuritis,pneumonia, and ophthalmic infections (EXAMPLES 4 and 11).

Representative uses of the subject methods of the invention include usein conjunction with a vaccine to enhance the immune response to thevaccine and provide a higher Ievel of immunity and/or a prolongedanamnestic response. The subject R'-Glu-Trp-R" compositions can beadministered prior to, simultaneously with, or following vaccination.Generally, the compositions will be administered prior to, orsimultaneously with, vaccination. Representative examples of usefulvaccines include viral vaccines, toxoids, meningococcal polysaccharidevaccine, diphtheria antitoxin, tetanus, tetanus immune globulin,pertussis vaccine, measles vaccine, mumps vaccine, rubella vaccine,PRP-D, Polysaccharide, PRP-OMP, rabies immune globulin, BCG vaccine,cholera vaccine, plague vaccine, smallpox vaccine, vaccine immuneglobulin, typhoid vaccine, yellow fever vaccine, varicella-zoster immuneglobulin, botulism antitoxin trivalent, and cytomegalovirus immuneglobulin.

Representative uses of the subject methods of the invention in treatingbacterial infections in a combined treatment with an anti-infectiveagent such as an antibiotic, e.g. one or more penicillins,cephalosporins, aminoglycosides, macrolides, sulfa compounds,fluoroquinolones, or tetracyclines. In the latter case, the subjectR'-Glu-Trp-R" compositions may be administered simultaneously with, orshortly before or after the chosen anti-infective agent. Representativepharmaceuticals that may be administered in conjunction with the subjectdipeptide compositions in the methods of the invention include (but arenot limited to) anti-infective agents such as: penicillin G, penicillinV, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillion,ampicillin, amoxicillin, bacampicillin, cyclacillin, carbenicillinindanyl, ticarcillin, mezlocillin, piperacillin, cephalothin, cefazolin,cephapirin, cephradine, cephalexin, cefadroxil, cefamandole nafate,cefuroxime, cefonicid, ceforanide, cefaclor, cefoxitin, cefotetan,cefinetazole , cefataxime, ceftizoxime, ceftriaxone, ceftazidime,cefoperazone, moxalactam, cefixime, erythromycin, stearate,ethylsuccinate, estolate, lactobionate, gluceptate, azithromycin,clarithromycin oxytetracycline, demeclocycline, doxycycline,minocycline, amikacin sulfate, gentamicin sulfate, intrathecal,kanamycin sulfate, netilmicin sulfate, streptomycin sulfate, tobramycinsulfate, neomycin sulfate, sulfadiazine, sulfmethizole, sulfisoxazole,sulfisoxazole acetyl, sulfamethoxazole, trisulfapyrimidines,phenazopyridine, erythromycin ethylsuccinate, Trimethoprim,Ciprofloxacin, Ciprofloxacin hydrochloride, enoxacin, Lomefloxacinhydrochloride, Norfloxacin, Ofloxacin, vancomycin hydrochloride,teicoplanin, rifampin, metronidazole, metronidazole hydrochloride,polmyxins, bacitracin, methenamine, methenamine hippurate, methenaminemandelate, nitrofurantoin, phenazopyridine hydrochloride, silvernitrate, acetic acid, Domeboro solution, m-cresyl acetate, Colymycin Sotic, cortisporin, tridesilon, ciclopiroxolamine, clioquinol,griseofulvin, fulvicin, grisactin, grisactin ultra, grifulvin V,halaprogin, pyrithione zinc, selenium sulfide, tolnaftate, undecylenicacid, naftfine, terbinafind, imidazole, econazole, ketoconazole,miconaxole nitrate, Monistat-Derm, oxiconazole nitrate, sulconazolenitrate, bis-triazoles, intraconazole, amphotericin B, nystatin,mycolstatin, nilstat, butoconazole, clotrimazole, tioconazold,fluconazole, intraconazole, terconazole, nystatin, mycostatin, O-VStatin, cantharidin, interferon-α_(2a), interferon-α₃, intralesional,podophyllin resin, podofilox, salicylic acid, benzylbenzoate,crotamiton, lindane, malathion, permethrin, phrethrins, piperonylbutoxide, sulfur, isoniazid, pyrazinamide, ethambutol, capreomycinsulfate, cycloserine, ethambutol hydrochloride, ethionamide,clofazimine, dapsone, ethionamide, itraconazole, potassium iodideflucytosine, chloroquine phosphate, hydroxychloroquine phosphate,chloroquine hydrochloride, quinine sulfate, pyrimethamine/sulfadoxine,mefloquine, quinidine gluconate, dilozanide furoate, eflornithinehydrochloride, furazolidone, iodoquinol, melarsoprol, metronidazole,nifurtimox, paramomycin sulfate, pentamidine isethionate, primaquinephosphate, quinine sulfate, sodium stibogluconate, meglumineantimoniate, trimetrexate glucuronate, pyrimethamine, albendazole,diethyclcarbamazine citrate, ivermectin, mebendazole, metrifonate,niclosamide, oxamniquine, pyrantel pamoate, suramin sodium,thiabendazole, cytarabine, idoxuridine, trifluridine, vidarabine,acyclovir, Zidovudine, ribavirin, bromovinyldeoxyuridine,fluoroiodoaracytosine, amantadine, acemannan, amphotericin B methyl,Ampligen, castanospermine, soluble CD₄, dextran sulfate,dideoxycytidine, dideoxyinosine, didihydrodideoxythymidine, foscarnetsodium, fusidic acid, HPA-23, isoprinosine, penicillamine, peptide T,ribavirin, rifabutin, interferon-α_(2b), didanosine, zalcitabine, andthe like.

Representative uses of the subject methods of the invention in acombined regimen for treating a mycobacterial infection includesadministering the subject R'-Glu-Trp-R" composition and at least oneanti-infective agent to a subject in need thereof The mycobacterialinfection may be localized or generalized, e.g., pulmonary anddisseminated lesions of Mytobacterium leprae (e.g., as disclosed inEXAMPLE 18, below) or Mycobacterium tuberculosis (e.g., as disclosed inEXAMPLE 21, below). The subject methods may prove particularly useful intreating mycobacterial infection which are resistant to antibiotics. Theanti-infective agent will generally be administered according to itsstandard dosage schedule. For example, treatment of Mycobacteriumtuberculosis infections may comprise administering the dipeptides (orcorresponding polymeric or cyclic forms) to the host in conjunction withstandard therapy, such as isoniazid, rifampin, ethambutol, streptomycin,or pyrazinamide. These agents will generally be administered accordingto treatment protocols of the World Health Organization (Geneva,Switzerland) or Centers for Disease Control (Atlanta, Ga.). Treatment ofMycobacterium leprae infections may include administration of acomposition of the present invention, as well as dapsone, rifampin,clofazimine, or ethionamide according to standard protocols as suggestedby the World Health Organization (Geneva, Switzerland) or NationalHansen's Disease Center (Carville, La.).

Representative uses of the subject methods in treating subjects withmycotic infections include treatments for patients having candidiasis(systemic or mucocutaneous), aspergillosis, blastomycosis,chromoblastomycosis, coccidio-mycosis, cryptococcosis, histoplasmosis,mucormycosis, paracoccidiodomycosis, pseudallescheriasis, orsporotichosis. The subject treatments for mycotic infections includecombined therapeutic regimens in which the subject composition isadministered with an anti-fungal agent, e.g., amphotericin B,flucytosine, ketoconazole, fluconazole, itraconazole, and the like, to asubject in need thereof.

Representative uses of the subject methods of the invention in treatmentof viral infections include treatment of subjects having infections withHIV-1, HIV-2, cytomegalovirus, herpes viruses, HTLV-I, HTLV-II, hogcholera virus, distemper virus, feline sarcoma virus, hepatitis viruses,influenza virus, and Dengue virus. The subject methods include those inwhich the R'-Glu-Trp-R" compositions are administered in a combinedtreatment regimen with an anti-viral agent such as an interferon-α,interferon-β, interferon-γ, interferon α_(2b), cytarabine, acyclovir,idoxuridine, vidarabine, ganciclovir, Zidovudine, ribavirin,bromovinyldeoxyuridine, amantidine, foscamet, dideoxyinosine,dideoxycytidine, azidothymidine, and the like. Illustrative treatmentsof subjects having viral infections are disclosed in the Examplessection, as follows: patients having herpes infections (EXAMPLE 8);Dengue fever virus infections (EXAMPLE 20); influenza infections,vaccination, prophylaxis, and treatment of symptoms (EXAMPLE 4);hepatitis B virus infections (EXAMPLE 25); and HIV-1 infections (EXAMPLE5).

Representative uses of the subject methods for treating subjects withparasitic infections includes patients having leishmaniasis,pneumocystis infections, giardiasis, trypanosomiasis, malaria,toxoplasmosis, coccidiosis, trichomoniasis, trichinosis, clonorchiasis,echinococcosis, dirofilariasis, and the like. The subject treatmentmethods with R'-Glu-Trp-R" are exemplified by methods disclosed below inwhich L-Glu-L-Trp compositions were administered in a combined treatmentregimen with an anti-parasitic agent, e.g., with a quinine derivative ina patient with malaria (as illustrated in EXAMPLE 19).

Treatment Methods

Illustrative uses of the subject methods of the invention in treatingclinical symptoms of immunodeficiency diseases, and symptoms of variousbacterial, viral, or inflammatory diseases states are illustrated in theExamples section. The dose, route, and duration of administration of theR'-Glu-Trp-R" compositions can be determined by a skilled practitioneraccording to recognized and accepted methods of clinical practice, suchas monitoring patient response to therapy (i.e., clinical indicia ofdisease) and/or laboratory test results (i.e., laboratory indicia ofdisease). In addition, methods for administration of R'-Glu-Trp-R" aredisclosed in greater detail in the EXAMPLES section. It is routine inthe practice of medicine for a physician to examine and treat patientswith various infectious diseases, autoimmune diseases, orimmunodeficiency diseases. Examples of infectious diseases include innerear infections, sinus infections, urinary tract infections, pneumonia,bacterial endocarditis, osteomyelitis and the like. Examples ofautoimmune diseases include systemic lupus erythematosus, rheumatoidarthritis, multiple sclerosis, type I diabetes, myasthenia gravis,Sjogren's syndrome, and the like. Examples of immunodeficiency diseasesleading to immunocompromised hosts include primary and secondaryimmunodeficiency syndromes such as Wiscott-Aldrich syndrome andHIV-infection. Some syndromes are less severe than others. For examplebacterial endocarditis can be life threatening, whereas inner earinfections are rarely life threatening. In deciding whether to use thesubject methods of the invention in combination therapy, e.g., withantibiotics or other biologically active agents (e.g., interferons orinterleukins) to treat the particular syndrome, a physician will usuallybase the decision on knowledge of whether opportunistic bacterial,viral, or parasitic infections are commonly involved in subjectsyndrome. Such knowledge has been accumulated over decades, and isreported in the medical literature as well as medical texts. The timingof when to start the subject methods in combination or single agenttherapy rests on the physician's clinical judgement. For lifethreatening infections, the physician will usually obtain the specimensfor culture (e.g., for a throat or blood culture) and begin treatmentwith what has become standard empiric antibiotic therapy. Empirictherapy is a therapy designed to treat the most common or likelycausative agent based on historic, demographic, and epidemiologicinformation. Empiric therapy may often include use of multipletherapeutic agents (e.g., more than one antibiotic and/or broad spectrumantibiotics) designed to cover a wide range of therapeuticpossibilities. When laboratory test data are available (commonly about48 hours for bacteriology or immunology tests and 3 to 7 days forvirology tests) the choice of therapy may be adjusted to moreparticularly treat the disease (e.g., based on the results of in vitroantibiotic sensitivity testing). Because treatment of clinical syndromesis very often initiated empirically (before the causative agent orunderlying condition has been identified), it would be very difficult ifnot impossible to test clinically a new therapeutic agent or method forone particular bacteria (e.g., staphylococcus). Rather, a newtherapeutic method must be tested for a particular clinical syndrome. Itis important to note that, for any given infectious syndrome, severaldifferent infectious agents or underlying conditions may be potentialcausative agents of the disease. In the art of pharmaceutical drugdevelopment, preclinical studies of a therapy evaluate the therapy'seffects on not just one infectious agent or condition, but on multipleagents or conditions of interest. The results of the various (sometimesequivocal) studies are weighed as to the benefits and risks of theparticular therapy given the medical knowledge of the risks associatedwith a particular disease. It is common that not all patients with asyndrome are cured by a single therapy, but instead, that a subset ofpatients may exist wherein the therapy has a positive and favorableresult. Examples of clinical syndromes in which subsets of patients mayfind favorable outcomes from the subject therapies of the invention aredisclosed in the following several paragraphs.

Representative uses of the subject methods for treating animmunocompromised subject may alleviate symptoms such as susceptibilityto one or more opportunistic bacterial or viral infections includingthose caused by Pneumocystis, cytomegalovirus, herpes virus,Staphylococcus, and the like. In one embodiment, a patient isadministered an amount of R'-Glu-Trp-R" sufficient to induce an increasein either the total number of lymphocytes, T-lymphocytes, CD4⁺-ymphocytes, CD8⁺ -lymphocytes, and the like. Illustrative examples ofimmunocompromised patients who may benefit from the subject treatmentmethods are provided in the Examples section including: i) patients withcancer following radiation therapy, i.e., breast cancer patients andpatients with thoracic cavity tumors and other cancers after radiationtherapy (EXAMPLE 3, Protocols A-C); ii) patients having occupationalradiation exposure (EXAMPLE 12, Protocols A and B); and, iii) patientsfollowing adult thymectomy EXAMPLE 24). Illustrative examples of otherimmunocompromised patients who may benefit from the subject treatmentmethods include patients having one or more temporary immune defectsresulting from i) chronic bacterial infections with M. leprae or M.tuberculosis (EXAMPLES 18 and 21, respectively); ii) chronic hepatitis Bvirus infections (EXAMPLE 25, Protocol B); patients havingpyelonephritis and prostatitis (EXAMPLE 17); osteomyelitis (EXAMPLE 16);and psoriasis EXAMPLE 6).

Representative uses of the subject methods for treating a subject alsoinclude treatments designed to promote wound healing, e.g., epithelial,mucosal, and bone defects. Illustrative examples of patients who maybenefit from the subject treatment methods include patients havingduodenal or gastric ulcers (EXAMPLE 23), bone fractures (EXAMPLE 16);and, epithelial defects including those resulting from leprosy (EXAMPLE18), psoriasis, burns and frostbite (EXAMPLE 6). Representative uses ofthe subject methods for treating a subject also include treatmentsdesigned to reduce systemic toxicity, e.g., manifested by symptoms offever, chills, migraine headaches, muscle aches and the like.Illustrative examples of patients who may benefit from the subjecttreatment methods include patients having jaundice with systemictoxicity (EXAMPLE 25); toxemia associated with complications ofpregnancy (EXAMPLE 7); and, fever, chills, and systemic toxicityassociated with malarial infection or Dengue virus infection (EXAMPLES19 and 20, respectively).

Representative uses of the subject methods for treating a subject alsoinclude treatments of patients with allergic disease states, includingpatients with acute respiratory allergic reactions, urticaria, hives,hay fever, asthma, and the like (EXAMPLES 14 and 22, below).

Representative uses of the subject methods for treating a subject alsoinclude treatments of patients with graft-versus-host disease. Bonemarrow transplant patients may be treated according to the methods ofthe invention to lessen the immunoreactivity of the transplantedimmunologically-active cells against the host tissue.

Representative uses of the subject methods for treating a subject alsoinclude treatments of patients with dental caries, gingivitis, andperiodontitis. Illustrative uses of the subject methods in treatment ofpatients with gingivitis and periapical granulomas is disclosed inEXAMPLE 9 (Protocols A and C).

The pharmaceutical compositions of the invention are intended forparenteral, topical, subcutaneous, intramuscular, intrathecal, oral,intranasal, or local administration for prophylactic and/or therapeutictreatment. Preferably, the compositions of the present invention areadministered intramuscularly or intranasally. The subject R'-Glu-Trp-R"compositions herein have the advantage of providing the desired effectsat very low dosage levels and without toxicity. Thus, a purpose oftherapy in an acute setting may be to rapidly increase the concentrationof R'-Glu-Trp-R" in a tissue, e.g., by bolus intravenous injection orinfusion. Alternatively, in other cases it may desirable to deliverR'-Glu-Trp-R" over a longer period of time.

Pharmaceutical Compositions

The subject compositions containing R'-Glu-Trp-R" may be formulated in amanner that allows absorption into the blood stream. The presentcompositions are immunomodulators that induce changes at the cellularlevel that subsequently effect changes in cellular processes that nolonger are dependent on the presence of the composition. So, in manyinstances it has been observed that the effects of the peptide arelong-lasting, i.e., for weeks to months, despite the rather rapiddegradation of the peptide, e.g., within minutes or hours. Although thesubject R'-Glu-Trp-R" compounds are themselves water-soluble at the lowconcentrations in which they are usually employed, they are preferablyused in the form of their acid or alkaline salts formed withpharmaceutically acceptable agents, e.g., acetic, citric, maleic,succinic acid, sodium, potassium, ammonium, or zinc (as disclosed ingreater detail below). Freely-soluble salts of the subject R'-Glu-Trp-R"compositions may also be converted to salts of low solubility in bodyfluids, e.g., by modification with a slightly water-solublepharmaceutically acceptable salt like tannic or palmoic acid, or byinclusion in a time-release formulation with covalently coupling to alarger carrier, or inclusion in timed-release capsule and the like.

The active dipeptide ingredient of the pharmaceutical preparationsaccording to the present invention may be used as a free peptide or inthe form of a water soluble pharmaceutically acceptable salt, such as asodium, potassium, ammonium or zinc salt. It will be understood that thedipeptide may be administered with other active ingredients whichindependently impart an activity to the composition, such as,antibiotics, interferon, anesthetics, and the like. Pharmaceuticallyacceptable salts may be conveniently prepared from EW dipeptide oranalogs by conventional methods. Thus, such salts may be, for example,prepared by treating EW dipeptide with an aqueous solution of thedesired pharmaceutically acceptable metallic hydroxide or other metallicbase and evaporating the resulting solution to dryness, preferably underreduced pressure in a nitrogen atmosphere. Alternatively, a solution ofEW dipeptide may be mixed with an alkoxide to the desired metal, and thesolution subsequently evaporated to dryness. The pharmaceuticallyacceptable hydroxides, bases, and alkoxides include those with cationsfor this purpose, including (but not limited to), potassium, sodium,ammonium, calcium, and magnesium. Other representative pharmaceuticallyacceptable salts include hydrochloride, hydrobromide, sulfate,bisulfate, acetate, oxalate, valarate, oleate, laurate, borate,benzoate, lactate, phosphate, tosulate, citrate, maleate, furmarate,succinate, tartrate, and the like.

For parenteral administration, the present invention providespharmaceutical preparations which comprise a solution of atryptophan-containing dipeptide, polymeric, multimeric, cyclic orderivative form thereof, dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, andthe like, including proteins and/or glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with a sterile aqueous solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc. Itmay be desirable to stabilize EW dipeptides, analogs, receptorfragments, and the like to increase their shelf life and pharmacokinetichalf-life. Shelf life stability is improved by adding excipients suchas: a) hydrophobic agents (e.g., glycerol); b) sugars (e.g., sucrose,mannose, sorbitol, rhamnose, xylose); c) complex carbohydrates (e.g.,lactose); and/or d) bacteriostatic agents. Pharmacokinetic half-lifes ofpeptides are modified by coupling to carrier peptides, polypeptides, andcarbohydrates by chemical derivatization (e.g., by coupling side chainor N- or C-terminal residues), or chemically altering the amino acid toanother amino acid (as above). Pharmacokinetic half-lifes andpharmacodynamics may also be modified by: a) encapsulation (e.g., inliposomes); b) controlling the degree of hydration (e.g., by controllingthe extent and type of glycosylation of the peptide); and, c)controlling the electrostatic charge and hydrophobicity of the peptide.

A presently most preferred formulation according to the instantinvention is a solution for intramuscular injection containing about0.001 to 0.01% by weight (0.0001-0.001 mg/kg body weight, or 10-100 μgactive ingredient per 1 mL solvent). The pharmaceutically acceptablevehicle for this preferred injection form may be any pharmaceuticallyacceptable solvent such as 0.9% aqueous sodium chloride, distilledwater, Novocaine solution, Ringer's solution, glucose solution, and thelike. The dipeptide containing compositions according to the presentinvention may be administered with a compatible pharmaceutical suitablefor parenteral administration (e.g., intravenous, subcutaneous, orintramuscular). The preparations may be subjected to conventionalpharmaceutical operations, such as sterilization, and may containadjuvants, such as preservatives, stabilizers, wetting agents and thelike. The peptides in the present compositions are typicallybiologically active at a dose of about 0.5 μg/kg to about 10 μg/kg,preferably about 1 μg/kg to about 5 gg/kg. The concentration of thepeptides in these pharmaceutical compositions can vary widely, i.e.,from about 0.001% to as much as 15 or 20% by weight and will be selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected. When utilizedintramuscularly, the injection solution has the active ingredient in atherapeutically effective immunopotentiating amount of about 0.001 to0.01% by weight. If prepared in the form of a tablet, capsule orsuppository, it is preferred that the active ingredient be present in anamount of about 0.1 mg per tablet, suppository or capsule. In such form,the capsule, suppository or tablet may also contain other conventionalexcipients and vehicles such as fillers, starch, glucose, etc. Intopical preparations, the peptides are generally contained in urea-basedemollients, petroleum-based ointments, and the like at concentrations ofabout 0.1 to 10,000 parts per million, preferably about 1 to 1000 partsper million, and most preferably about 10 to 100 parts per million.Actual methods for preparing parenterally, orally, and topicallyadministrable compounds will be known or apparent to those skilled inthe art and are described in detail in, for example, Remington'sPharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa.(1985), which is incorporated herein by reference.

Intramuscular and intranasal routes are preferred for administration ofthe subject R'-Glu-Trp-R" compositions. One preferred dosage of thesubject composition for intramuscular administration is about 50 μg to100 μg per dose for adults (for a 300 μg to 1000 μg total treatmenttherapy); for infants up to 1 year old about 10 μg per dose, for infants1 to 3 years old about 10 μg to 20 μg per dose; for infants 4 to 6 yearsold about 20 μg to 30 μg per dose, for children 7 to 14 years old about50 μg per dose. All of the foregoing dosages are useful for a treatmentof 3 to 10 days, depending upon the immunodeficiency level. Thetreatment may be repeated as needed, usually within 1 to 6 months. Inanother preferred embodiment, a treatment dose of about 10 μg/kg toabout 1 mg/kg of a pharmaceutical preparation of R'-Glu-Trp-R" isadministered to a subject daily over a period of about 6 days to about10 days, but optionally at the discretion of the attending physician,for up to about 30 days. In one preferred course of therapy,R'-Glu-Trp-R" is administered im daily at a dosage of 1-100 μg/kg for5-7 days, followed by a 1-6 month resting period before repeating thesame injection regimen.

The subject methods include those in which the R'-Glu-Trp-R" areadministered by injection, e.g., by parenteral, intramuscular,intradermal, subcutaneous, and intraperitoneal injection. In a mostpreferred embodiment, the treatment dose is sufficient to increase anumber or percentage of CD2⁺ or Fc-receptor-bearing lymphocytes inperipheral blood or in a reticuloendothelial tissue, or to increase anumber of immune cells in an inflammatory infiltrate, or to increase aproportion of phagocytically active cells in an inflammatory infiltrate.

For embodiments where the subject compositions of the invention isintended for treatment of an infectious disease in a subject, an amountis administered that is sufficient to cure or at least partially arrestthe disease and its complications. An amount adequate to effect thistherapeutic result in more than 50% of subjects so treated is defined asa "therapeutically effective dose." Amounts effective for this use willdepend on the severity of the infection or disease and the weight andgeneral state of the patient being treated, but generally range fromabout 0.1 mg/kg to about 5000 mg/kg host body weight of R'-Glu-Trp-R"dipeptide per day, more commonly about 0.2 mg/kg to about 1000 mg/kghost body weight of dipeptide per day, usually about 0.5 μg/kg to about100 μg/kg host body per day, more usually about 0.75 μg/kg to about 20μg/kg host body weight per day, and preferably about 1 μg/kg to about 10μg/kg host body weight per day. Maintenance dosages over a prolongedperiod of time may be adjusted as necessary. Typical total daily dosesare about 50 to 100 μg in adults, about 50 μg in children 7-14 years ofage, about 20-30 μg in children 4 to 6 years of age, about 10-20 μg inchildren 1-3 years of age and about 10 μg in children less than one yearof age. The compositions may be administered once daily or more often asdesired. Treatment of acute conditions generally will occur over about3-10 days. Treatment of chronic conditions or prophylactic treatmentshave the same course, but can be repeated after as long as about 1-6months or longer. In some instances, it may be desirable to administerthe compositions intermittently on a daily basis for periods of about 2to about 20 days, preferably about 3 to about 14 days, more preferablyabout 4 to about 10 days which are repeated at least about 15 days,preferably about 20 days or as much as about 1 to 6 months or more.

The route of delivery of R'-Glu-Trp-R" dipeptides and the like isdetermined by the disease and the site where treatment is required. Fortopical application it may be desirable to apply the dipeptide, analogs,agonists, and antagonists at the local site ((e.g., by placing a needleinto the tissue at that site) or by placing an impregnated bandageduring surgery); while for more advanced diseases it may be desirable toadminister the compositions systemically. For other indications, theR'-Glu-Trp-R" dipeptides, analogs, agonists, antagonists, derivativesand the like may be delivered by intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, and intradermal injection, aswell as, by intrabronchial instillation (e.g., with a nebulizer),transdermal delivery (e.g., with a lipid-soluble carrier in a skinpatch), or gastrointestinal delivery (e.g., with a capsule or tablet).

In general, the acid addition salts of the subject R'-Glu-Trp-R", e.g.,L-Glu-L-Lys, compositions with pharmaceutically acceptable acids will bebiologically equivalent to the subject compositions themselves.

The preferred therapeutic compositions, inocula, routes, and dosageswill vary with the clinical indication. For intramuscular injection, theinoculum is typically prepared from a dried peptide (or peptideconjugate) by suspending the peptide in a physiologically acceptablediluent such as water, saline, or phosphate-buffered saline. Somevariation in dosage will necessarily occur depending upon the conditionof the patient being treated, and the physician will, in any event,determine the appropriate dose for the individual patient. The effectiveamount of peptide per unit dose depends, among other things, on the bodyweight, physiology, and chosen inoculation regimen. A unit dose ofpeptide refers to the weight of peptide without the weight of carrier(when carrier is used). An effective treatment will be achieved when theconcentration of R'-Glu-Trp-R" dipeptide, e.g., L-Glu-L-Trp, at a tissuesite in the microenvironment of the cells approaches a concentration of10⁻⁵ M to 10⁻⁹ M. Skilled practitioners can make use of clinical andlaboratory indicia to monitor patient response to the subject therapyand adjust the dosage accordingly. Since the pharmacokinetics andpharmacodynamics of R'-Glu-Trp-R" dipeptides, agonists, antagonists, andthe like will vary in different patients, a most preferred method forachieving a therapeutic concentration in a tissue is to graduallyescalate the dosage and monitor the clinical and laboratory indicia. Theinitial dose, for such an escalating dosage regimen of therapy, willdepend upon the route of administration. For intravenous administration,of R'-Glu-Trp-R" dipeptide with an approximate molecular weight of 200to 400 daltons, an initial dosage of approximately 0.1 mg/kg body weightis administered and the dosage is escalated at 1 0-fold increases inconcentration for each interval of the escalating dosage regimen.

For prophylactic uses against opportunistic infections inimmunodeficient or immunodepressed patients, the intramuscular and/orintranasal single daily dose for adults may be from about 50 to 10 μf,and for children about 10 μg to 50 μg per dose for treatment over 3 to 5days.

For treatment of burns, frost bite, or other wounds, including chronicapical periodontitis, the dipeptide may be applied in about 100 μg dosesas a paste or other suitable medium.

For ophthalmology, such as for treatment of infectious eye diseases, thedipeptide may be applied in single daily dosages of about 10 μg (over 4to 10 days) or as installations into the conjunctival cavity at about 5μg twice daily over about 4 to 5 days.

The dipeptide may be injected intramuscularly in an injection solutionhaving a therapeutically effective immunomodulatory amount of about0.001 to 0.01% by weight of the subject R'-Glu-Trp-R" composition. Ifpresented in the form of a tablet, capsule or suppository, it ispreferred that the active ingredient be present in an amount of about0.1 mg per tablet, suppository or capsule. If presented in such form,the capsule, suppository or tablet may also contain other conventionalexcipients and vehicles such as fillers, starch, glucose, etc.

Synthesis of R'-Glu-Trp-R"

Conveniently, the subject R'-Glu-Trp-R" dipeptide is synthesized by anyof a number of automated techniques that are now commonly available.Generally speaking, these techniques involve stepwise synthesis bysuccessive additions of amino acids to produce progressively largermolecules. The amino acids are linked together by condensation betweenthe carboxyl group of one amino acid and the amino group of anotheramino acid to form a peptide bond. To control these reactions, it isnecessary to block the amino group of one amino acid and the carboxylgroup of the other. The blocking groups should be selected for easyremoval without adversely affecting the peptides, i.e., by racemizationor by hydrolysis ofthe formed peptide bonds. Amino acids with carboxyl-groups (e.g., Asp, Glu) or hydroxyl- groups (e.g., Ser, homoserine, andTyr) also require blocking prior to condensation. A wide variety ofprocedures exist for synthesis of peptides, solid-phase synthesisusually being preferred. In this procedure an amino acid is bound to aresin particle, and the peptide generated in a stepwise manner bysuccessive additions of protected amino acids to the growing chain.Modifications of the technique described by Merrifield are commonly used(Merrifield, R. B., J. Am. Chem. Soc. 96:2989-2993 (1964)). In anexemplary automated solid-phase method, peptides are synthesized byloading the carboxy- terminal amino acid onto an organic linker (e.g.,PAM, 4-oxymethyl phenylacetamidomethyl) covalently attached to aninsoluble polystyrene resin that is cross-linked with divinyl benzene.Blocking with t-Boc is used to protect the terminal amine, and hydroxyl-and carboxyl- groups are commonly blocked with O-benzyl groups.Synthesis is accomplished in an automated peptide synthesizer (AppliedBiosystems, Foster City, Calif., e.g., Model 430-A). Following synthesisthe product may be removed from the resin and blocking groups removedusing hydrofluoric acid or trifluoromethyl sulfonic acid according toestablished methods (Bergot, B. J. & S. N. McCurdy, Applied BiosystemsBulletin (1987)). A routine synthesis can produce 0.5 mmole ofpeptide-resin. The yield following cleavage and purification isapproximately 60 to 70%. For example, an amino and side chain protectedderivative of an activated ester of Glx is reacted with side-groupprotected Trp, attached to the solid phase at its C-terminus. Afterelimination of the alpha-amino protecting group, the peptide may becleaved from the solid phase or another amino acid added in a similarfashion. Additional amino acids are serially added in a similar fashion.The peptides are then cleaved by acid that also typically removesprotecting groups. The peptides may then be isolated and lyophilized andstored for future use. Suitable techniques of peptide synthesis aredescribed in detail in Stewart & Young, SOLID PHASE PEPDE SYNThESIS, 2dedition, Pierce Chemical Company, 1984; and Tam, et al., J. Am. Chem.Soc. 105:6442 (1983), both of which are incorporated herein byreference. Purification of the product peptides is accomplished, forexample, by crystallizing the peptide from an organic solvent such asmethyl-butyl ether, followed by dissolving in distilled water, anddialysis (if the molecular weight of the peptide is greater than about500 daltons), thin layer chromatography, gel chromatography,lyophilization, or reverse HPLC (e.g., using a C18 column with 0.1%trifluoroacetic acid and acetonitrile as solvents) if the molecularweight of the peptide less than 500 daltons. Purified peptide islyophilized is stored in a dry state until use. A representativeR'-Glu-Trp-R" pharmaceutical preparation is the purified dipeptideL-Glu-L-Trp, which comprises a white powder (if lyophilized; otherwise,it is crystalline), soluble in water, DMF; insoluble in chloroform andether. alpha22_(D) =+12.6; C=0.5 H₂ O. R_(f) =0.65 (butanol:aceticacid:water=3:1:1). UV (275±5 nm, max). NMR (500 MHz): 0.001 mol/l of thepeptide solution, Trp (3.17; 3.37; 4.57; 7.16; 7.24; 7.71; 7.49); Glu(1.90; 1.96; 2.21; 3.72)!.

Typically an amino and side chain protected derivative of an activatedester of glutamic acid is reacted with protected L-tryptophan. Afterelimination of the protecting groups and conventional purification, suchas by thin layer or GL chromatography, the peptide may be purified suchas by, lyophilization, gel purification, and the like.

Pharmaceutically Acceptable Carriers

The compounds may be administered alone or formulated withpharmaceutically acceptable carriers, in either single or multipledoses. Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solutions, and various nontoxic organicsolvents. Pharmaceutical compositions can be formed by combiningR'-Glu-Trp-R" dipeptide with a pharmaceutically acceptable carrier andan optional antibiotic. The subject combination therapeutic agents canthen readily be administered in a variety of dosage forms such astablets, lozenges, syrups, injectable solutions, and the like.Combination therapeutic agents may also include R'-Glu-Trp-R" dipeptide,e.g., L-Glu-L-Trp, in the same unit dosage form. Pharmaceutical carrierscan, if desired, contain additional ingredients such as flavorings,binders, excipients, and the like. Thus, for purposes of oraladministration, tablets containing various excipients such as sodiumcitrate, calcium carbonate, and calcium phosphate may be employed alongwith various disintegrants such as starch, and preferably potato ortapioca starch, alginic acid, and certain complex silicates, togetherwith binding agents such as polyvinylpyrolidone, sucrose, gelatin, andacacia. Additionally, lubricating agents, such as magnesium stearate,sodium lauryl sulfate, and talc are often useful for tableting purposes.Solid compositions of a similar type may also be employed as fillers insalt and hard-filled gelatin capsules. Preferred materials for thispurpose include lactose or milk sugar and high molecular weightpolyethylene glycols. When aqueous suspensions of elixirs are desiredfor oral administration, the essential active R'-Glu-Trp-R" dipeptidetherein may be combined with various sweetening or flavoring agents,colored matter or dyes, and if desired, emulsifying or suspendingagents, together with diluents such as water, ethanol, propylene glycol,glycerin, and combinations thereof For parenteral administration,solutions of R'-Glu-Trp-R", analogs, or receptor fragments in sesame orpeanut oil or in aqueous polypropylene glycol may be employed, as wellas sterile aqueous saline solutions of the corresponding water solublepharmaceutically acceptable metal salts previously described. Such anaqueous solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal injection. The sterileaqueous media employed are all readily obtainable by standard techniqueswell known to those skilled in the art. Additionally, it is possible toadminister the aforesaid compounds topically (e.g., through a placedcatheter) using an appropriate solution suitable for the purpose athand.

The following examples are provided to further elucidate the invention,but are not intended to restrict the invention in scope or spirit in anyway. Experimental animal trials (disclosed herein in EXAMPLES 26-36)determined the effects of L-Glu-L-Trp at dosages of about 10 μg/kg toabout 1 mg/kg on different lymphocyte subpopulations and lymphoid organsin vivo. The results of the studies disclosed in EXAMPLES 26-36 aresummarized in TABLES A-D. The results indicate that in an experimentalanimal model, treatments with L-Glu-L-Trp at a dosage of about 0.1 mg/kgto about 1 mg/kg was effective i) to increase CD2⁺ lymphocytes inperipheral blood by about 3-fold; ii) to increase Fc receptor bearinglymphoid cells in spleen and bone marrow (BM) by about 1.3-fold andabout 1.7-fold, respectively (TABLE A); iii) to mobilize cells from thespleen and thymus into peripheral blood with resultant reduction inspleen and thymic mass (TABLE B); and, iv) to increase neutrophilinfiltration into tissues in response to an inflammatory agent by about2-fold with about a 1.7-fold increase in phagocytically active cells inthe infiltrate (TABLE C).

                  TABLE A                                                         ______________________________________                                        CD2.sup.+  and Fc-Receptor Lymphocytes in Treated Guinea Pigs                             CD2+        Fc-receptor+                                          Lymphoid    L--Glu--L--Trp                                                                            L--Glu--L--Trp                                        Organ       Treatment   Treatment                                             ______________________________________                                        Thymus      ns          ns                                                    Spleen      ns          1.7 X ↑ (p < 0.05)                              LN          32% ↓ (p < 0.05)                                                                   ns                                                    BM          50% ↓ (p < 0.05)                                                                   1.3 X ↑ (p < 0.05)                              PBL         3 X ↑ (p < 0.05)                                                                    ns                                                    ______________________________________                                         *ns, no statistically significant change; CD2+ lymphocytes (ERFC), result     presented in EXAMPLE 28 (TABLE 51), below; Fcreceptor lymphocytes (EARFC)     results presented in EXAMPLE 29 (TABLE 52), below.                       

                  TABLE B                                                         ______________________________________                                        Splenic and Thymic Mass in Treated Mice*                                      Lymphoid     L--Glu--L--Trp                                                   Organ        Treatment                                                        ______________________________________                                        Thymus       44% ↓ (p < 0.01)                                          Spleen       15% ↓ (p < 0.05)                                          ______________________________________                                         *ns, no statistically significant change; results presented in EXAMPLE 30     (TABLE 54), below.                                                       

                  TABLE C                                                         ______________________________________                                        Neutrophils Induced by a Sterile Inflammatory Agent in Treated Mice*          Neutrophil     L--Glu--L--Trp                                                 Index          Treatment                                                      ______________________________________                                        Total cells X10.sup.6                                                                          2 X ↑ (p < 0.05)                                       Phagocytic cells (%)                                                                         1.7 X ↑ (p < 0.05)                                       Phagocytic Index                                                                             ns                                                             ______________________________________                                         *ns, no statistically significant change; results presented in EXAMPLE 30     (TABLES 58-59), below.                                                   

The results shown in the Examples section, disclose effects ofL-Glu-L-Trp treatments on lymphocytes, macrophages and neutrophils(EXAMPLES 26-31)as follows: i) activating anti-microbial activity ofresident macrophages (EXAMPLE 30); ii) promoting of neutrophilinfiltration into tissues in response to inflammatory agents (EXAMPLE30); iii) stimulating formation of antibody forming cells in the spleenEXAMPLE 31); iv) enhancing responsiveness of T-lymphocytes (EXAMPLES26-30); v) mobilizing lymphocytes from lymph nodes (and from tissues)into peripheral blood and spleen (EXAMPLE 30); and, vi) mobilizinglymphocytes from bone marrow and thymus into peripheral blood EXAMPLE30). The observed respective immunologic effects of L-Glu-L-Trp arecompatible with the interpretation that the subject treatments of theinvention heighten the state of innate immunity to infection in asubject so treated.

In certain therapeutic uses it may prove useful to monitor dipeptidelevels in bodily fluids while escalating the dose delivered to thepatient. The representative R'-Glu-Trp-R" dipeptide, e.g., L-Glu-L-Trp,is non-mutagenic in vitro, non-toxic to human and guinea pig lymphocytesin vitro, non-toxic when in injected intraperitoneally into guinea pigsand mice, and without adverse effects in human preclinical trials, asdisclosed in the EXAMPLES.

Analogs of R'-Glu-Trp-R"

The amino acid sequence of the R'-Glu-Trp-R" dipeptide permitspreparation of appropriate nucleotide sequences (e.g., by standardtechniques), and incorporation of these sequences into bacterial, yeast,and insect plasmid DNA, as well as into mammalian cell viral vectors(e.g., retroviral vectors.) Expression systems that may be used toproduce the peptides of the invention include prokaryotic, eukaryotic,yeast, and insect cells. It is presently believed likely that EW is acytomedine released from hydrolysis of tissue polypeptides, at a ratehomeostatically determined by tissue pH and enzymatic activity in thetissues. The present disclosure serves as a usefuil basis forconstructing derivatized and; covalently modified EW analogs,antagonists, and the like which can be screened and tested forbiological activities. For example, EW may be used for preparation of ananalog that is, e.g., a) covalently modified by adenylation,methylation, acylation, phosphbrylation, uridylation, fatty-acylation,glycosylation, and the like; b) a sterioisomer of an L-Glu-L-Trp, e.g.,replacing a D- for an L-sterioisomer, and the like; c) a derivative ofEW, wherein one amino acid is substituted for another of likeproperties, i.e., a neutral nonpolar amino acid for another neutralnonpolar (e.g., W replaced by S, T, Y, N, Q, or C), an acidic amino acidfor another acid (e.g., E replaced by D), or a basic amino acid foranother basic (e.g., K replaced R or H); d) a chemically modified formof EW, e.g., a C-terminal (or gamma-carboxyl) group modified to acarbonyl, or an N-terminal group modified to an amide, or N- orC-terminal extension with Sar or gamma-amino butyric acid (GABA); e) achemically derivatized form of EW, e.g., covalent coupling of the IMpeptide to a larger peptide (or polypeptide) carrier, or an N- orC-terminal extended peptide; or, f) replacing one amino acid withanother of slightly different properties, e.g., changing thehydrophobicity of the dipeptide. Alternative methods for identifying thesubject R'-X-Trp-R" dipeptides of the invention are disclosed in U.S.Ser. No. 08/370,838, incorporated herein by reference.

"Analog" is intended to mean a chemical compound according to FormulaI-III, that mimics or improves on the electronic, steric, hydrophobic,and 3-dimensional space filling requirements of the groups in an EWdipeptide that are involved in binding to a cellular EW-receptor (e.g.,alpha side chain residues, and/or the amino and carboxyl groups inL-Glu-L-Trp). Representative analogs include chemical mimetic compoundsthat are capable of antagonizing binding of EW to a ligand-receptor,i.e., antagonists (as defined below), and other ligands that are capableof binding to a ligand receptor and exerting effects similar to EW,i.e., agonists (as defined below).

"Agonist" as used herein means a chemically modified EW, or organicchemical molecule according to Formula I-III, that is capable ofspatially conforming to the molecular space filled by an EW ligand andthat is further capable of combining with the subject ligand receptorsto initiate an action that is initiated by EW following binding to theirspecific ligand receptor(s) on cells in vitro or in vivo. Representativeexamples of actions initiated by EW may be: i) upregulation oflymphocyte cell surface determinants such as CD2, CD4, CD28, CD45, CD58,CD59, LFA 1, ICAM 1, ICAM 2, ICAM 3, and the like; ii) macrophageactivation; iii) stimulating (or inhibiting) lymphocyte blastogenesis inresponse to antigen (or niitdgen), iv) stimulating release ofinterleukins from lymphocytes (e.g., IL2, IL4, IL5, IL6, IL10, IFNγ,TGFβ and the like); v) mobilization of intracellular calcium (e.g.,through membrane calcium channels) and cAMP; vi) stimulating cellinteractions between T-helper lymphocytes, B-lymphocytes and antigenpresenting cells in initiation of primary and secondary humoral(antibody-mediated) immune responses; vii) stimulation of macrophages toincrease their antibacterial and cytotoxic activities to microbial andmammalian target cells, and the like. Agonists possess binding affinityfor ligand-receptor(s) and intrinsic activity for inducing the immuneeffects that are induced when EW bind to their ligand receptor. Ascreening assay for identifying a candidate R'-Glu-Trp-R" agonist, mayconsist of the following steps: (a) synthesizing a chemical mimeticcompound, e.g., a compound of Formula I, II, or III; (b) introducing thecompound into a T-cell rosette assay as a test article; and (c)determining that the test article has an activity substantially similarto the activity of L-Glu-L-Trp in the T-cell rosette assay.

"Antagonist" as used herein means a chemical molecule according toFormula I-III, that spacially conforms to the molecular space filled byEW and is further capable of combining with the subject ligandreceptor(s), as set forth above, to inhibit, neutralize, impede orreverse, at least in part, an action initiated following binding of EWto the subject ligand receptor on a lymphocyte or a macrophage. Ascreening assay for identifying a candidate R'-Glu-Trp-R" antagonistanalog, may consist of the following steps: (a) synthesizing a chemicalmimetic compound, e.g., a compound of Formula I, II, or III; (b)introducing the analog into a T-cell rosette assay as a test article;and (c) determining that the test article inhibits the activity ofL-Glu-L-Trp in the T-cell rosette assay.

Assays for R'-Glu-Trp-R" and its Analogs

Screening assays for analogs, e.g., antagonists and agonists, mayinclude comparative testing of the subject test articles for biologicalactivities exhibited by L-Glu-L-Trp in vivo in experimental animal modelsystems (e.g., Examples 1-44). Alternatively, it may prove convenient tomonitor activities of the test agents in in vitro assays whereinL-Glu-L-Trp have been shown to exhibit biologic activity, e.g., changesin calcium flux or cyclic nucleotide levels, and changes inintracellular second messenger pathways triggered by the addition ofL-Glu-L-Trp to lymphocytes. Biological activity of a compound accordingto Formula II or Formula III of Example 45 may for example be determinedby testing for second messenger pathways triggered by receptor bindingthe subject compound. Second messenger pathways may be monitored, e.g.,by testing intracellular levels of Ca⁺⁺, cAMP, cGMP, adenyl cyclase,tyrosine kinase, guanylate cyclase, protein kinase, phosphorylase A,protein kinase C; or, cellular release of interleukins, arachidonic acidmetabolites, prostaglandins, and the like.

The present disclosure of significant biological effects of EWdipeptides on immune cells also serves as basis for isolating cellsurface receptors binding EW, and for identifying other ligands inbiological fluids which may bind to the subject receptors.

"Ligand" as used herein refers to a compound that is capable of fillingthe three-dimensional space in a receptor binding site so thatelectrostatic repulsive forces are minimized, electrostatic attractiveforces are maximized, and hydrophobic and hydrogen bonding forces aremaximized. L-Glu-L-Trp is a representative ligand. Ligands bind to theirspecific receptors in a specific and saturable manner. A candidateligand may be tested to determine whether it is an analog byradiolabeling the candidate ligand and then testing whether its bindingto lymphocytes is specifically and competitively inhibited in thepresence of an excess (e.g., 1000-fold molar excess) of unlabeledL-Glu-L-Trp. Alternatively, a test may be conducted to determine whetherthe candidate ligand inhibits binding of radiolabeled L-Glu-L-Trp tolymphocytes. A positive test result in either assay may be taken as anindication that the candidate ligand is an analog. A negative testresult in either assay is indeterminant.

"Ligand receptor" is used herein to refer to a receptor capable ofbinding a ligand, according to the definition above, in a specific andsaturable manner. In an illustrative assay for identifying a ligandreceptor in a sample of cells, a labeled ligand (e.g., a radiolabeled orbiotin-labeled L-Glu-L-Trp) at a concentration from within the range of0.1 nM to 10 mM, is incubated at room temperature, 37° C., and 4° C.with an aliquot of about 10⁵ -10⁷ cells. Following the incubation, thecells are washed (e.g., by centrifugation through an isobutylpthalate orsucrose cushion) and the amount of labeled ligand associated with thecell pellet is determined (e.g., by quantifying radioactivity orreacting the sample with enzymatically-labeled avidin, washing to removeunbound avidin, and then adding substrate to visualize theenzyme-bound-avidin-biotin receptor complex). The data obtained in thismanner may be plotted in a Scatchard plot from which an associationconstant (K_(a)) of the receptor for the ligand may be determined. Theassociation constant for binding of a ligand (e.g., EW dipeptide) isabout 0.01 nM to about 1 mK and most preferably about 1 nM to about 50μM. Affinity chromatography e.g., on solid phase resins containingimmobilized EW dipeptide (EW-resin), may prove useful for preparingsubstantially purified preparations of the subject EW receptors andtheir fragments. Purified EW receptors and receptor fragments may beuseful as pharmacological inhibitors, antagonists, and agonists of EWdipeptide binding in vivo. Receptor fragments may for example beisolated by affinity chromatography of crude cell and tissuehydrolysates on EW-resin columns, or alternatively, by binding thereceptor to an EW-resin and then treating with one or more selectedproteases to create EW receptor fragments which are either eluted fromthe column (i.e., non-ligand binding portions of the receptor), orremain ligand-binding portions which remain bound to the resin. Theligand-binding portions of the receptor may be eluted from the resineither by raising the salt concentration (e.g., to 1-4 M NaCl, or 0.5-2M guanidine HCl) to negate electrostatic binding interactions, or bydropping the pH to induce a conformational change in the receptorfragment. The amino acid sequence of the EW receptor is convenientlydetermined by automated amino acid sequencing, and the sequence may beused to construct synthetic peptides, and nucleotide probes (e.g.,degenerate probes) for cloning the subject EW receptor.

"Ligand-receptor fragments" is a term used to refer to portions of thesubject ligand receptor that are smaller in size than a ligand receptorisolated from a natural source, e.g., tissue, biological fluids and thelike. Fragments may be prepared from a ligand receptor isolated from atissue and then subjected to proteolytic degradation or treatment with achemical such as cyanogen bromide. In the latter case, the subjectfragments of the receptor are conveniently purified before use, e.g., byreverse-phase HPLC or immunoaffinity chromatography. Alternatively,fragments of the ligand receptor may be prepared by expressing a portionof a nucleotide sequence of a genomic or cDNA clone capable ofexpressing the subject ligand receptor, e.g., a portion of ligandreceptor nucleotide sequence in an expression plasmid or vectorintroduced into a cell, wherein the cell manufactures the subjectreceptor fragment and the fragment can be purified (as above). Fragmentsof the subject ligand receptor may be soluble in biological fluids andaqueous solutions and may bind EW with a greater or lesser K_(D) than acomplete (non-degraded) ligand receptor.

"Substantially purified" as used herein with respect to peptides refersto a preparation from a natural source that contains either a "peptide"(i.e., intended to mean about 2 to about 18 amino acids), a ligandaccording to criteria set forth above, or a ligand receptor or receptorfragment that is: i) enriched greater than about 50-fold from itsconcentration in a natural source material, and ii) contains less than5% peptide or polypeptide impurities detectable by reverse-phase HPLC,SDS-PAGE, or immunoassay.

Inhibitors of R'-Glu-Trp-R"

The disclosure herein of biological activities of EW dipeptides providesa basis for identifying and isolating proteins that eitherproteolytically inactivate EW dipeptides, or that alter binding of thesedipeptides to EW receptors. "EW-peptidase" are used herein to refer todipeptidylpeptidases capable of catalyzing hydrolysis of theglutamyl-tryptophane peptide bond. The subject enzymes inactivate EWpeptides and render them biologically inactive. EW-peptidases mayconveniently be purified from tissues and cells using conventionalpurification methods (e.g., affinity chromatography on EW-resin columns,as above, alternatively, ion exchange and/or molecular sievechromatography using FPLC or HPLC systems). Purification may bemonitored using calorimetric or fluorescent EW peptide substrates, andprotease test assays. The subject protease test assays may, in turn,prove useful for identifying natural and synthetic inhibitors ofEW-peptidases. The subject EW dipeptidylpeptidase inhibitors may havebiological effects similar to EW, since they may decrease proteolyticdegradation of EW dipeptide and thus increase the biological half-lifeof a natural EW dipeptide arising endogenously within a tissue.

The present disclosure further provides a basis for diagnosticimmunoassays, antibody reagents and the like for measuring levels of EWin biological fluids. While the subject EW dipeptides are relativelynonimmunogenic, (falling into the class of low molecular weighthaptens), antibodies may be induced in experimental animals when theyare conjugated to a carrier (e.g., KLH). Polyclonal and monoclonalantibodies to EW find uses in a variety of immunoassay formats fordiagnostic monitoring the levels of the subject dipeptides in bodilyfluids in health and disease. Representative immunoassay formatsinclude: enzyme linked immunoabsorbent assays (ELISA), radioimmunoassays(RIA), fluorescence immunoassay (FIA), time-resolved fluorescence assays(TRF), and cascade assay formats that are routinely used in the art forincreasing low-end sensitivity of assays. Individuals with high levelsof EW dipeptide may be at decreased relative risk for infectiousdisease, while those with decreased levels may be at an increasedrelative risk. The EW dipeptide can be used as a positive and a negativecontrol in the subject diagnostic assays. The subject assays may beassembled for use in a reagent immunoassay test kit. The antibodiesdisclosed herein specific for EW may be useful in a variety ofcompetitive and non-competitive direct and indirect immunoassay formatsas will be apparent to skilled artisans. The level of EW in a biologicalsample is commonly determined by comparison with positive and negativecontrols and assay calibrators.

The term "biological fluids" is used herein to mean tissue fluids (suchas joint fluid, cerebrospinal fluid and the like), plasma and serum,fluids in body cavities (i.e., peritoneal fluid, lung lavage fluid,urogenital mucus secretions, and the like), urine, feces, sputum, sweat,and the like.

While not wishing to be tied to any particular molecular mechanisms ofaction, cytomedines are believed to interact with cellular receptors.Traditionally, cellular receptors have been believed to bind specificligands with a specific affinity. Binding (or non-binding) of the ligandto the cellular receptor is believed to induce certain cellularfunctions. It is further believed that cytomedines interact with thesecellular receptors. In addition to the ligand-bindiyng site, however,the receptors apparently have separate cytomedine-binding sites. It isbelieved that receptor-cytomedine binding can alter the conformation ofthe ligand-binding site so as to increase the affinity of the receptorfor the ligand, thereby altering the response ofthe cell to a particularconcentration of ligand within the microenvironment of the cell.Different cytomedines and different cytomedine concentrations can havevarying effects on the binding affinity between the receptor and theligand. Situations may exist in which cytomedine ligands interact withcellular receptors that have secondary binding sites for molecules otherthan the cytomedine, and in this case, the cytomedine-receptorinteraction may alter the binding for the other molecule. For example,when the dipeptide Glu-Trp interacts with T cells, there is an increasein receptor binding affinity for CD48, CD58, or CD59, and anaccompanying increase of intracellular cAMP concentrations in the cells.Increased intracellular cAMP in turn activates intracellular proteinkinase activity.

Protein kinase activity is an important factor in the signaltransduction pathway for activating transcription and translation ofproteins necessary for lymphocyte activation and progression of the celldivision cycle from G₀ /G₁ into mitosis.

Tryptophane containing dipeptides (e.g., EW, DW, NW, AW, QW, IW, SW, TW,SW, TW, GW, HEW, HW, LW, VW, EWEW, GEW, EGW, EWKHG, EWKKHG,EW-NH-NH-GHK-NH₂, Ac-L-Glu-L-Trp-OH, Suc-EW, Cpr-EW, But-EW,L-Glu-D-Trp, D-Glu-L-Trp, D-Glu-D-Trp, RKDVW, RKEVW, RKEWY, RKEW, KEWY,KEVW, KEW, GWY, pEW, and the like) may constitute improved deliveryvehicles for the amino acid tryptophan. Tryptophan is convertible bytissue hydroxylase enzymes, e.g., liver phenylalanine hydroxylase, into5-hydroxy-tryptamine (i.e., serotonin) and by mono-amino oxidase enzymesinto 5-hydroxy-indol-acetic acid in the pathway to melatonin. Serotoninis capable of binding to beta-adrenergic receptors, adrenoreceptors,serotonergic receptors, e.g., on endothelial cells, vascular andbronchial smooth muscle, neural cells, platelets, lymphocytes, CD 4⁺-lymphocytes, and myocardial cells. Metabolically modified EW, e.g.,modified to glutamyl-serotonin, may be rapidly hydrolyzed to releaseserotonin into tissues and blood.

While not wishing to be tied to any particular mechanism of action, itis believed likely that the subject tryptophan-containing peptidesreversibly associate with specific cellular EW receptors, one examplebeing provided by the T-lymphocyte E-rosette receptor defined as the"CD2" cell surface determinant. It is believed likely that binding of EWdipeptide to CD2 (and other EW receptors) trigger: i) conformationalchanges in the receptor that signal up-regulation of adenylate cyclaseand increased intracellular cAMP, and; ii) increased affinity of the CD2receptors for binding of EW dipeptides. It is presently believed likelythat L-Glu-L-Trp exerts its immunomodulatory effects by binding toEW-receptors and stimulating increased expression and/or affiniity ofCD2 molecules on T-lymphocytes and thymocytes. It is considered likelythat increases CD2 expression or binding affinity. Intracellularmechanisms triggered by dipeptide binding to the receptor may mediate:(a) changes in lymphocyte tissue distribution, ie., mobilization of oneor more subsets from lymphoid tissues into peripheral blood, (b)activation of one or more lymphocyte subsets; and/or (c) down- orup-regulation of the functional activity of one or more lymphocytesubsets. The changes induced at a cellular and molecular level by atryptophane-containing dipeptide may result in immune deviation, whereinan immune response (i.e., cellular or humoral) directed toward oneantigen is down-regulated and a response to a different antigen isup-regulated. As disclosed in the Examples section, increasedT-lymphocyte counts in peripheral blood is a visible manifestation ofthe mobilization from lymphoid tissues induced by treatment with EWdipeptides. Increased cytokine production, e.g. as assayed by leukocytemigration inhibition response (LMIR), is another particularmanifestation of activated T-lymphocytes. Activation of mononuclearphagocytes (e.g., monocytes) is manifested by increased phagocyticactivity, e.g., phagocytosis of latex or staphylococci, and activationof granulocytes is manifested as increased cell numbers in inflammatoryexudates as well as increased phagocytic activity. Effects oftryptophane-containing dipeptides on immunoresponsiveness of animals andman are disclosed in the Examples which follow. It is consideredpossible that the CD2 receptor on lymphocytes may bind serotonin.

EXAMPLE 1 Lack of Mutagenicity and Toxicity of L-Glu-L-Trp

Note that the Materials and Methods used in Examples 1-34, below, appearimmediately following Example 46, i.e., at the end of the Examplessection immediately before the citations.

Protocol A

Mutagenicity: On peripheral blood of human volunteers in vitro. Cellcultures were incubated and treated. As can be seen from Table 1, after24 hrs. incubation at concentrations of 1 μg/mL and 100 μg/mL, there wasstatistically no mutagenic effect in these cultures.

                  TABLE 1                                                         ______________________________________                                        Analysis of Possible Chromosomal Damage in                                    Human Peripheral Blood Lymphocytes                                                             Metaphases         Level of                                           Number of                                                                             chromosome                                                                              Index of mutagenic                                          analyzed                                                                              aberrations                                                                             reliability*                                                                           effect                                    Dose       metaphases                                                                              #      %    (P)    (numbers)                             ______________________________________                                        Control    1000      15     1.5  --     --                                    L--Glu--L--Trp -                                                                         1000      15     1.5  >0.05  0                                     1 μg/ml                                                                    L--Glu--L--Trp -                                                                         1000      16     1.6  >0.05  0                                     100 μg/ml                                                                  ______________________________________                                         *p > 0.05, not statistically significant difference from control cell         preparations.                                                            

Protocol B Acute Toxicity Studies

Summary: L-Glu-L-Trp when injected im at dosages calculated to be about10,000-times the therapeutic dosage were found to be non toxic in mice,guinea pigs, chickens, and dogs as determined by monitoring generalcondition, behavior, movements, cardiac and respiratory physiology, andgross pathology.

Protocol C Chronic Toxicity Studies

Summary: L-Glu-L-Trp when injected daily as a single im or iv for aperiod of 30 days was without adverse effects as determined bymonitoring behavior, feeding, body weight, coat condition, mucousmembranes, red and white cell blood counts, cardiac and respiratoryphysiology, liver and kidney function, and gross pathology. Kidneyfunction was determnined by evaluation of diuresis after water-loading;for certain other experiments animals were sacrificed and examined after10, 20, 30, and 60 days.

EXAMPLE 2 Accidental Radiation Induced Immunodeficiency: Chernobyl

Background: On Apr. 26, 1986 the largest nuclear catastrophe of our timetook place at 1:24 a.m. local time, destroying the fourth reactor of theChernobyl nuclear power plant (NPP). The reactor and its core werereportedly destroyed by an explosion causing two radioactive jetemissions of Iodine¹³¹ followed by Cesium¹³⁷ and gamma emissions(Milhaud, G., Biomedicine & Pharmacology 45(6):219-220 (1991)). Theaccident reportedly cause the deaths of 31 workers and firemen whointervened to bring the installation back under control, 203 individualswere hospitalized with acute radiation sickness, and more than 135,000persons were evacuated from the 30 km zone immediately adjacent to theNPP. Initial optimism that there would be no residual after affects,were not justified. Studies of individuals exposed to radiation at theChernobyl NPP have reported doses of whole body exposure at 0.1-0.5 Gyfor individuals involved in the clean-up; 4-9 Gy for power plant workers(Yarilyn, A. A., et al., Intl. J. Radiation Biology 63(4):519-528(1993)); and a range of 0.1-12.5 Gy for accidental exposure of differentindividuals in the 135,000 inhabitants within the 30 km zone surroundingthe NPP (Barkhudarov, R. M., Vestnik Akademii Meditsinskikh Nauk.SSSR8:3-9 (1991)); Gruzdev, et al., Radiobiologiia 32(1):3-18 (1992))Russian physicians treating the exposed individuals reported acorrelation between the numbers of lymphocytes in peripheral blood andthe severity of the acute radiation sickness, with an initial drop inthe average numbers of lymphocytes to 50% of normal by day 3-6post-exposure (Suvorova, L.A., et al., Radiobiologiia 31 291-6 (1991)).Decreased lymphocyte counts are reportedly correlated with the severityof radiation sickness (Hirsh, E. F., et al, World J. Surgery 16(5):918-923 (1992)). Hematological data have also been used to construct anempirical dose curve for gamma radiation exposure at Chernobyl andvalues reported for 127 of the former residents range from 0.5 Gy to 12Gy (Konchalovskii, M. V., et al., Meditsinskai Radiologiia 36(1): 29-33(1991)). For reference, 10 Gy is a 100% bone marrow lethal dose ofradiation in mice as determined 30 days post-exposure (Hendrickson, F.R, et al., CLINCAL ONCOLOGY, eds: Holleb, A. I., et al., Amer. CancerSoc., Atlanta, Ga., p. 42 (1992))). Human exposure to 2 Gy of whole bodyirradiation daily for 2 days (4 Gy total), has been reported to resultin decreased erythrocyte and granulocyte precursors in bone marrow.Daily exposure to 2 Gy whole body irradiation for 5 days (10 Gy total)has been reported to result in a total absence of undifferentiated anddifferentiated cellular forms and blast cells in the bone marrow withmaximal depression of peripheral blood lymphocytes counts being evidentat 10-15 days and cell counts approaching only 40% of normal.Reportedly, recovery generally occurs after 3-6 months. (Mettler, F. A.,et al, MEDICAL EFFECTS OF IONIZING RADIATION, Grune & Stratton Inc.,N.Y.; p. 165 (1985)).

Approximately 120,000 former Chernobyl residents are currentlyreportedly being followed to determine long term effects of radiationexposure. In patients suffering from acute radiation sickness during theaccident, immune defects have been reported three to five years laterwith decreased circulating T- and B-lymphocytes and increased levels ofserum autoantibodies (Iarilin, A.A., et al., Radiobiologiia 32: 771-778(1992)); Beliakov, I. M., et al., Radiobiologiia 32((30): 349-356(1992)); decreased antibacterial immunity (Bidnenko, et al., ZhunalMikrobiologii Epidemiologii I Immunologii 1: 33-36 (1992)); chromosomalaberrations in peripheral blood lymphocytes (Bochkov, N. P., et al.,Meditsinskaia Radiologiia 36(7): 50-52 (1991)); elevated numbers oflarge undifferentiated cells in peripheral blood, increased neutrophilcounts, and reduced numbers of lymphocytes and basophils (Liubchenko, etal., Laboratornoe Delo 8: 47-51 (1991)); increased morbidity from solidtumors (Behar, A., et al. J. Environ. Pathol. Tox. & Oncology10(6):281-285 (1991)); and, decreased monocyte phagocytic activity andhumoral immunity (Iakovlev, N. I., et al., Akusherstvo I Ginekologiya11: 42-45 (1991)); Ivanov, A. A., et al., Akushersivo I Ginekologiya11:42-45 (1991)); Ivanov, A. A., et al., GematologiyaI Transfuziologiya36(12):20-22 (1991)).

In geographic regions adjacent to Chernobyl, population epidemiologistsreported increased rates of respiratory virus infection (in Kiev;Vozianov, A. F., et al., Vrachebnoe Delo 3: 14-17 (1991)), and increasedtuberculosis mortality (in Byelorussian SSR; Kalechits, O. M., et al.,Problemy Tuberkuleza 11: 14-16 (1990); Dvoirin, M. S., et al., ProblemyTuberkuleza 11: 12-14 (1990)). Five years after the accident,experimental rats exposed for 30 days to ground radiation in the 30 kmChernobyl disaster zone reportedly received a cumulative body dose of 57rads (1 Gy=100 rads) and exhibited aberrations in bone marrow metaphasesand peripheral blood leukocyte counts (Izmozherova, et al.,Radiobiologiia 32(4): 493-9 (1992)). It has been suggested that exposureto sublethal levels of radiation may cause premature aging ofT-lymphocytes with lessened tumor surveilance activity (Davila, D. R.,et al., Int. J. Radiation Biol. 611: 123-133 (1992)); Sasaki, H., etal., J. Radiation Biol. 32 (suppl.): 310-326 (1991)). CD4⁺ T-lymphocytesare reportedly more radiosensitive than CD8⁺ (DeRuysscher, D., et al.,Eur. J Cancer 28A(10): 1729-1734 (1992)); Ceschia, T., et al.,Radiologia Medica 81(4): 532-536 (1991)).

The chief methods of therapy for individuals exposed to radiation in theChernobyl zone were reported to be administration of antimicrobial drugsand fresh donor platelets (i.e., "Conventional Therapy", in ProtocolsA-E, below). Stem cell transplantation using allogeneic bone marrowreportedly showed limited efficacy (Baranov, A. E., et al.,Meditsinskaia Radiologiia 36((3): 29-32 (1991)).

Summary Overview of Protocols A-E, below: In all, 348 Chernobylresidents and workers were examined (20-50 years of age; 320 men and 28women). The state of the immune systems in these individuals wasdetermined in the field using peripheral blood samples androsette-forming lymphocyte methods. In certain studies, patients wereexamined during hospitalization and T- and B-lymphocyte populations weredetermined using a flow cytometer and monoclonal antibodies specific forlymphocyte cell surface markers. Radiation exposure levels for thedifferent patients were determined using dosimetry data obtained by themilitary in Chernobyl using a D-2-P dosimeter. The time course of theimmunodepression in these subjects is summarized as follows: subjects inProtocol A, evaluated within the first few weeks after evacuation fromChernobyl showed peripheral blood leukocyte and lymphocyte counts thatwere only 60% of normal ("Before", TABLE 2). By 2 months (Protocol B)the lymphocyte counts in patients presenting for therapy had fallen toabout 40% of normal ("Before, TABLE 3), but by six months (Protocols C;D) improvement was noted, i.e., to 76% of normal ("Before", TABLE 5).Changes in total lymphocyte counts over the latter 6 month period weremirrored by increased numbers of CD4⁺ lymphocytes (i.e., from 10% at 4-5months (TABLE 4) to 43% at 6 months (TABLE 5); and CD8⁺ T-lymphocytes(i.e., from 39% at 4-5 months to 71% at 6 months).

Immunomodulator therapy was initiated in 1986 with Thymalin, (a mixtureof thymic peptides, the preparation of which was disclosed by some ofthe instant inventors in DE 3,421,789, Morozov, V. G., et al., (1985)).Treatments were administered to patients presenting in the clinic withinthe first few weeks after the Chernobyl NPP accident, and again at 2months, 4-5 months, and 6 months (Protocols A-D, below). Thymalin wasadministered prophylactically to a small group of workers whoparticipated in sealing the roof of reactor III (Protocol E). Inaddition, the immunomodulatory synthetic peptide L-Glu-L-Trp wasadministered to certain patients at three years post-exposure (i.e.,Protocol E, below).

Treatments with Thymalin were administered as 5 daily injection of 10mg. Treatments with L-Glu-L-Trp were administered as 5 daily iminjections of 100 μg each. The effects of the treatments on leukocyteswere monitored in the week following treatment by collecting samples ofperipheral blood from the different patients.

Protocol A: Early Values

The 42 patients described below, were tested for blood parameters thefirst few days to weeks after exposure to 0.2-0.5 Gy of environmentalradiation in Chernobyl ("Before") and then again after a 5 days ofThymalin therapy (daily doses of 10 mg im; "After"). A control group of34 Chernobyl subjects received conventional therapy (i.e.,antihistamines, multivitamins, and treatments for symptoms). Themajority of the subjects in this trial protocol, upon clinicalexamination, presented with complaints of weakness, scratchy throat,cough, acute eye pain, and a metallic taste in the mouth. Uponexamination, hyperemia of the fauces and conjunctiva was revealed. Nopathology was identified in the internal organs on gross examination. Itcan be seen from the data in TABLE 2 that a response to the Thymalintreatment was observed in the treatment population even at this earlytime after radiation exposure in Chernobyl.

                  TABLE 2                                                         ______________________________________                                        Thymalin Treatment of Chernobyl Subjects (X ± m):                          Treatments Initiated Shortly after Accidental Radiation Exposure                         Examination Group                                                               Healthy                                                          Laboratory   Normal    Accidental Radiation Exposure                          Indicia.sup.a                                                                              Controls  Before     After Thymalin                              ______________________________________                                        Leukocytes, abs                                                                            5.7 ± 0.3                                                                            3.8 ± 0.3*                                                                            6.4 ± 0.8**                              % Normal Value:                                                                            (100%)    (67%)      (112%)                                      Ratio Post-/Pre-Treat.sup.b :                                                              --        --         (1.68)                                      Lymphocytes, abs                                                                           1.91 ± 0.12                                                                          1.15 ± 0.14*                                                                          2.27 ± 0.16**                            % Normal Value:                                                                            (100%)    (60%)      (119%)                                      Ratio Post-/Pre-Treat:                                                                     --        --         (1.97)                                      CD2-DR+, %   30.8 ± 1.1                                                                           17.6 ± 2.0*                                                                           31 ± 3**                                 CD2-DR+, abs 0.59 ± 0.04                                                                          0.20 ± 0.03*                                                                          0.69 ± 0.08**                            % Normal Value:                                                                            (100%)    (34%)      (117%)                                      CD2, %       50.6 ± 1.6                                                                           47 ± 4  50.9 ± 2.4                               CD2, abs     0.98 ± 0.09                                                                          0.55 ± 0.08*                                                                          1.13 ± 0.07**                            % Normal Value:                                                                            (87%)     (56%)      (115%)                                      Ratio Post-/Pre-Treat:                                                                     --        --         (2.05)                                      E-RFC, %     29.7 ± 2.5                                                                           29.8 ± 2.6                                                                            23.4 ± 2.6                               Ratio Post-/Pre-Treat:                                                                     --        --         (0.79)                                      LMIR with ConA, %                                                                          66 ± 4 98 ± 9* 60 ± 7**                                 CD19 (C3-receptor+), %                                                                     22.8 ± 2.2                                                                           27.0 ± 2.8                                                                            30.5 ± 1.9                               CD19, abs    0.47 ± 0.03                                                                          0.30 ± 0.05*                                                                          0.68 ± 0.04**                            % Normal Value:                                                                            (100%)    (64%)      (145%)                                      Ratio Post-/Pre-Treat:                                                                     --        --         (2.26)                                      IgM, g/l     1.1 ± 0.4                                                                            0.51 ± 0.08*                                                                          0.58 ± 0.10*                             % Normal Value:                                                                            (100%)    (46%)      (53%)                                       IgG, g/l     10.1 ± 0.9                                                                           8.6 ± 1.3                                                                             9.2 ± 0.7                                % Normal Value:                                                                            (100%)    (85%)      (91%)                                       IgA, g/l     1.71 ± 0.16                                                                          2.07 ± 0.20                                                                           1.11 ± 0.09**                            % Normal Value:                                                                            (100%)    (121%)     (65%)                                       C3, g/l      0.57 ± 0.03                                                                          0.74 ± 0.07                                                                           0.68 ± 0.04                              ______________________________________                                         .sup.a abs, absolute cell no. × 10.sup.9 /L;                            .sup.b Post/Pre-Treat = Aftertreatment value/Beforetreatment value;           *statistically significant (p < 0.05) in comparison with the indices in       healthy normal subjects;                                                      **statistically significant (p < 0.05) in comparison with the data            obtained prior to L--Glu--L--Trp treatment;                                   LMIRleukocyte migration inhibition response; abscell concentration            presented as 10.sup.9 /L.                                                

The observed improvement in immune parameters (TABLE 2) was correlatedwith a material improvement in the ability of the treated subjects toperform work, and a reduction in the hyperemia of the mucosa.Non-treated subjects in the control group exhibited a continued declinein ability to perform work and reported headaches and insomnia.

Protocol B: Two Months of Therapy

Among the victims at the time of the Chernobyl accident, 1^(st) -3^(rd)degree acute radiation sickness was confirmed in 200 cases and, inaddition, still other subjects received in excess of 0.8 Gy withclinical signs of radiation trauma. Examination of individuals in thislatter group revealed indications of immune dysfunction. Twenty-threesuch adult patients, exposed to 1.0-3.0 Gy of radiation, were treated at2 months, and again at 6 months post-exposure. The patients received 10mg Thymalin daily for 5-10 consecutive days by intramuscular injection.Twelve normal healthy individuals were evaluated in a control group toestablish normal immune parameters. The results of the evaluation("Before") and treatment ("After") at 2 months are shown in TABLE 3.

                  TABLE 3                                                         ______________________________________                                        Treatment of Radiation-Induced Immunodeficiency: Treatments                   with Thymalin at Two Months Post-Radiation Exposure (X ± m)                           Examination Group                                                             Healthy Accidental Irradiation                                                  Normal               After Thymalin                              Indicia.sup.a                                                                              Control   Before     Treatment                                   ______________________________________                                        Leukocytes, abs                                                                            5.6 ± 0.8                                                                             3.5 ± 0.4*                                                                           5.0 ± 1.2**                              % Normal Value:                                                                            (100%)    (63%)      (89%)                                       Ratio Post-/Pre-Treat.sup.b :                                                              --        --         (1.43)                                      Lymphocytes, abs                                                                           1.98 ± 0.16                                                                           0.80 ± 0.24*                                                                         1.9 ± 0.4**                              % Normal Value:                                                                            (100%)    (40%)      (96%)                                       Ratio Post-/Pre-Treat:                                                                     --        --         (2.38)                                      CD2-DR+, %   35.8 ± 0.9                                                                           21 ± 4* 30.0 ± 1.2**                             CD2-DR+, abs 0.59 ± 0.04                                                                           0.16 ± 0.04*                                                                         0.55 ± 0.06*                             % Normal Value:                                                                            (100%)    (27%)      (93%)                                       CD2, %       49.3 ± 1.5                                                                           32 ± 7  48.7 ± 1.8**                             CD2, abs     0.98 ± 0.09                                                                           0.55 ± 0.08*                                                                         1.13 ± 0.08**                            % Normal Value:                                                                            (100%)    (56%)      (115%)                                      Ratio Post-/Pre-Treat:                                                                     --        --         (2.05)                                      E-RFC, %     30.2 ± 1.6                                                                           22.9 ± 1.9*                                                                           27.4 ± 2.4                               Ratio Post-/Pre-Treat:                                                                     --        --         (1.19)                                      LMI with ConA, %                                                                           65.0 ± 2.1                                                                           120 ± 17*                                                                             90 ± 10                                  CD19, %      22.0 ± 1.7                                                                           32 ± 3* 27 ± 4                                   CD19, abs    0.46 ± 0.03                                                                           0.26 ± 0.06*                                                                         0.51 ± 0.10**                            % Normal Value:                                                                            (100%)    (57%)      (110%)                                      Ratio Post-/Pre-Treat:                                                                     --        --         (1.96)                                      IgM, g/L     1.1 ± 0.4                                                                            0.87 ± 0.07                                                                           1.00 ± 0.10                              % Normal Value:                                                                            (100%)    (79%)      (91%)                                       IgG, g/L     11.1 ± 0.9                                                                           10.2 ± 2.0                                                                            10.0 ± 1.0                               % Normal Value:                                                                            (100%)    (92%)      (90%)                                       IgA, g/L     1.70 ± 0.10                                                                          1.5 ± 0.4                                                                             1.49 ± 0.19                              % Normal Value:                                                                            (100%)    (88%)      (88%)                                       ______________________________________                                         .sup.a abs, absolute cell no. × 10.sup.9 /L;                            .sup.b Post/Pre-Treat = Aftertreatment value/Beforetreatment value;           *statistically significant (p < 0.05) in comparison with the indices in       healthy normal subjects;                                                      **statistically significant (p < 0.05) in comparison with the data            obtained prior to L--Glu--L--Trp therapy;                                     LMIR--leukocyte migration inhibition response;                                RFC--rosetteforming cells.                                               

The results in TABLE 3 show a statistically significant increase incirculating leukocytes, lymphocytes, and CD2⁺ lymphocytes in peripheralblood following treatment with Thymalin. No change in CD19⁺ peripheralblood B-lymphocytes was observed. Simultaneous with the restoration ofimmune system parameters in peripheral blood, subjects in the Thymalintreated group exhibited a marked decline in symptoms associated with theasthenic syndrome. Clinical improvements induced by Thymalin weremaintained for 24 months post-therapy, after which the clinicalcondition of the patients again worsened. Control subjects receivingconventional treatment did not exhibit any statistically significantchange in immune parameters.

Protocol C: Therapy at 4 and 6 Months Post-Exposure

Acute radiation sickness (ARS) was manifest at 2-6 months in manypatients. Seven patients with ARS after exposure to 1.0-2.0 Gy ofradiation in Chemobyl were treated at 4-5 months post-exposure with100-150 mg Thymalin. daily for 10-15 days (1^(st) course) followed by 10mg Thymalin once a week for a period of 18 months (2^(nd) course).Immune parameters were evaluated before therapy, and immediatelyfollowing the first and second courses of treatment (TABLE 4 The resultspresented in TABLE 4 show significant increases in the number ofleukocytes, and CD2⁺ -DR⁺ - and CD3-lymphocytes after the first andsecond courses of therapy.

                  TABLE 4                                                         ______________________________________                                        Thymalin Therapy: 4-6 Months Post-Exposure (X ± m)                                    Time Interval of Examination                                                              After 1st   After the 2nd                                           Before    course of   course of                                  Indicia      Therapy   Thymalin    Thymalin                                   ______________________________________                                        Leukocytes, abs.sup.a                                                                      3.5 ± 0.5                                                                             4.7 ± 0.2*                                                                            5.5 ± 0.3*                              Ratio Post-/Pre-Treat.sup.b :                                                              (1.0)     (1.34)      (1.57)                                     Lymphocytes, abs                                                                           1.0 ± 0.5                                                                            1.5 ± 0.4                                                                              1.9 ± 0.5*                              Ratio Post-/Pre-Treat:                                                                     (1.0)     (1.5)       (1.9)                                      CD2-DR+, %   12.8 ± 2.6                                                                           22.3 ± 0.5                                                                             29 ± 3*                                 CD2-DR+, abs 0.13 ± 0.04                                                                           0.34 ± 0.05*                                                                          0.56 ± 0.08*                            Ratio Post-/Pre-Treat:                                                                     (1.0)     (2.6)       (4.3)                                      CD3, %       24 ± 3 35 ± 4*  46 ± 3*                                 CD3, abs     0.26 ± 0.05                                                                           0.49 ± 0.06*                                                                          0.89 ± 0.11*                            Ratio Post-/Pre-Treat:                                                                     (1.0)     (1.9)       (3.4)                                      CD4, %       7.1 ± 1.1                                                                            19.5 ± 1.7*                                                                            24.1 ± 1.5*                             CD4, abs     0.07 ± 0.01                                                                           0.28 ± 0.03*                                                                          0.45 ± 0.04*                            Ratio Post-/Pre-Treat:                                                                     (1.0)     (4.0)       (6.4)                                      CD8, %       17 ± 3 15.4 ± 2.3                                                                             22.3 ± 2.2*                             CD8, abs     0.16 ± 0.04                                                                          0.23 ± 0.03                                                                            0.40 ± 0.05*                            Ratio Post-/Pre-Treat:                                                                     (1.0)     (1.4)       (2.5)                                      CD19, %      12.2 ± 1.9                                                                           15.0 ± 2.8                                                                             21.1 ± 2.1*                             CD19, abs    0.14 ± 0.04                                                                          0.21 ± 0.06                                                                            0.39 ± 0.06*                            Ratio Post-/Pre-Treat:                                                                     (1.0)     (1.5)       (2.8)                                      ______________________________________                                         .sup.a statistically significant (p < 0.05) in comparison with the indice     prior to the first treatment;                                                 "a", abscell concentration presented as 10.sup.9 /L;                          "b", ratio = posttreatment value/pretreatment value.                     

Protocol D: 6 Months:

Periodic examinations of patients revealed a subset of Chernobylpatients who received 1.0-3.0 Gy of radiation in Chernobyl and whosubsequently exhibited great fluctuations in leukocyte counts. Thissubset of patients included 32 patients who were tested for bloodparameters at 6 months, and then treated with Thymalin at a dosage of 10mg daily for 10 consecutive days in the hopes of stabilizing immuneparameters. Twenty other patients served as a control group, and theywere treated using conventional methods. The results presented in TABLE5 show marked decreases in T- and B-lymphocyte subpopulations inpatients prior to therapy with reduced CD2-DR⁺ -, CD2³⁰ -, CD3⁺ -, andCD4⁺ -lymphocyte counts and decreased levels of B-lymphocytes withsurface immunoglobulin. Treatment with Thymalin resulted instatistically significant increases in the following lymphocytesubpopulations: CD2⁺ -DR⁺ -, CD2⁺ -, CD3⁺ -, CD4⁺ T-lymphocytes, and,CD19⁺ - and surface Ig⁺ -B-lymphocytes. The latter increases inlymphocyte subpopulations were not observed in peripheral blood samplesfrom patients treated in the control group with the conventionaltherapy. CD8⁺ -lymphocytes did not change reliably as a result of theThymalin treatment (TABLE 5).

                                      TABLE 5                                     __________________________________________________________________________    Thymalin Treatment of Acute Radiation Sickness:                               Treatments 6 Months After the Chernobyl Accident (X ± m)                             Normal Irradiated Subjects                                                    Healthy                                                                              Conventional                                                                         Thymalin Therapy:                                     Indices:  Controls                                                                             Therapy                                                                              Before After                                          __________________________________________________________________________    Lymphocytes, %                                                                          33.9 ± 1.2                                                                        32.9 ± 2.4                                                                        29.2 ± 2.0                                                                        30.0 ± 1.8                                  Lymphocytes, abs                                                                        1.96 ± 0.06                                                                        1.49 ± 0.14*                                                                     1.39 ± 0.13                                                                        1.52 ± 0.12*                               % Normal Value:                                                                         (100%) (76%)  (71%)  (78%)                                          Ratio Post-/Pre-Treat:                                                                  --     --     (0.9)  (1.02)                                         CD2, %    53.6 ± 1.9                                                                        38.7 ± 2.7*                                                                       32 ± 3*                                                                            49 ± 3**                                   CD2, abs  1.05 ± 0.05                                                                        0.56 ± 0.04*                                                                      0.44 ± 0.04*                                                                      0.75 ± 0.05**                              % Normal Value:                                                                         (100%) (53%)  (42%)  (71%)                                          Ratio Post-/Pre-Treat:                                                                  --     --     (0.79) (1.34)                                         CD2-DR+, %                                                                              30.8 ± 1.1                                                                        18.9 ± 1.6*                                                                       19.7 ± 1.2*                                                                        20.8 ± 1.6**                               CD2-DR+, abs                                                                            0.59 ± 0.04                                                                        0.30 ± 0.25*                                                                      0.28 ± 0.02*                                                                      0.31 ± 0.02**                              % Normal Value:                                                                         (100%) (51%)  (47%)  (53%)                                          CD3, %    55.6 ± 1.9                                                                        39.0 ± 2.4*                                                                       37 ± 5*                                                                            53.4 ± 1.8**                               CD3, abs  1.09 ± 0.08                                                                        0.58 ± 0.04*                                                                      0.51 ± 0.03*                                                                      0.82 ± 0.04**                              % Normal Value:                                                                         (100%) (53%)  (47%)  (75%)                                          CD4, %    35.3 ± 2.7                                                                        20.3 ± 1.3*                                                                       18.9 ± 1.3*                                                                        32.6 ± 1.4**                               CD4, abs  0.69 ± 0.05                                                                        0.30 ± 0.03*                                                                      0.26 ± 0.03*                                                                      0.50 ± 0.04**                              % Normal Value:                                                                         (100%) (43%)  (38%)  (72%)                                          Ratio Post-/Pre-Treat:                                                                  --     --     (0.9)  (.167)                                         CD8, %    21.3 ± 0.9                                                                        19.5 ± 1.5                                                                        17.5 ± 1.6                                                                        21.2 ± 1.8                                  CD8, abs  0.41 ± 0.03                                                                       0.29 ± 0.03                                                                       0.24 ± 0.03                                                                       0.32 ± 0.03                                 % Normal Value:                                                                         (100%) (71%)  (59%)  (78%)                                          Ratio Post-/Pre-Treat:                                                                  --     --     (0.8)  (1.10)                                         T4/T8     1.64 ± 0.12                                                                        1.04 ± 0.04*                                                                      1.08 ± 0.10*                                                                      1.54 ± 0.11**                              LMI       59.7 ± 1.7                                                                        106 ± 6*                                                                          107 ± 6*                                                                           72.7 ± 4.5**                               CD19, %   25.00 ± 0.12                                                                      18.2 ± 2.1*                                                                       23 ± 3                                                                            26.7 ± 2.1                                  CD19, abs 0.49 ± 0.04                                                                        0.27 ± 0.03*                                                                      0.31 ± 0.05*                                                                      0.41 ± 0.03**                              % Normal Value:                                                                         (100%) (55%)  (63%)  (84%)                                          Ratio Post-/Pre-Treat:                                                                  --     --     (1.15) (1.52)                                         B-Ig+, %  13.8 ± 1.2                                                                        15.8 ± 1.3                                                                        16.2 ± 1.7                                                                        19.0 ± 1.3                                  B-Ig+, abs                                                                              0.29 ± 0.02                                                                       0.23 ± 0.03                                                                       0.23 ± 0.04                                                                       0.29 ± 0.03                                 % Normal Value:                                                                         (100%) (79%)  (79%)  (100%)                                         Ratio Post-/Pre-Treat:                                                                  --     --     (1.0)  (1.26)                                         B-IgM+, % 6.4 ± 0.7                                                                         6.3 ± 0.8                                                                         5.4 ± 0.5                                                                         8.8 ± 0.7                                   B-IgM+, abs                                                                             0.12 ± 0.01                                                                        0.09 ± 0.01*                                                                      0.08 ± 0.01*                                                                     0.13 ± 0.02                                 B-IgG+, % 4.1 ± 0.5                                                                          7.8 ± 0.9*                                                                        7.1 ± 0.8*                                                                       6.4 ± 0.5                                   B-IgG+, abs                                                                             0.082 ± 0.008                                                                     0.098 ± 0.007                                                                     0.104 ± 0.008                                                                     0.100 ± 0.007                               B-IgA+, % 2.20 ± 0.20                                                                        1.80 ± 0.15*                                                                      1.70 ± 0.20*                                                                     1.8 ± 0.3                                   B-IgA+, abs                                                                             0.038 ± 0.004                                                                     0.033 ± 0.004                                                                     0.024 ± 0.003                                                                     0.030 ± 0.002                               IgM, g/L  1.15 ± 0.06                                                                       1.14 ± 0.08                                                                       1.20 ± 0.07                                                                       1.07 ± 0.09                                 % Normal Value:                                                                         (100%) (99%)  (104%) (93%)                                          IgG, g/L  11.5 ± 0.5                                                                        11.9 ± 1.0                                                                        11.7 ± 0.9                                                                        10.9 ± 1.1                                  % Normal Value:                                                                         (100%) (103%) (102%) (95%)                                          IgA, g/L  1.9 ± 1.0                                                                         1.6 ± 0.8                                                                         1.6 ± 0.8                                                                         1.8 ± 0.9                                   % Normal Value:                                                                         (100%) (84%)  (84%)  (95%)                                          __________________________________________________________________________     "a", abs cell concentration presented as 109/L; "b", Post/Pre-Treat Ratio     = posttreatment value/pretreatment value; *statistically significant (p <     0.05) vs. the indices in healthy people; **statistically significant (p <     0.05) vs. the data obtained before treatment; abs cell concentration          presented as 10.sup.9 /L; LMIleukocyte migration inhibition.             

Correlated with the observed improvements in immune parameters, clinicalimprovements were observed in the treated patients including a lack ofinfectious complications over the following two years of observation.Asthenic symptoms in 5 of the 7 treated patients were brought completelyunder control.

Protocol E: Prophylactic Administration Prior to Irradiation

A special investigation of the potential immunoprotective effects ofThymalin was assessed in a group of 20 volunteers who worked on the roofof the Chernobyl NPP reactor III. These subjects were given 10 mgThymalin daily on each of the three days they worked in the 30 km highradiation zone, with treatments commencing one day before the work onthe NPP. Twenty other volunteers were given glucose tablets, as acontrol. The volunteers received a dose of radiation equal to0.25+/-0.02 Gy over each of the 1-2 days they worked on the NPP. Immuneparameters were evaluated before administering Thymalin, and again onday 7 after the completion of the NPP work project. Volunteers whoreceived the placebo demonstrated changes in the number of CD2⁺ -,CD2-DR⁺ -, and CD8⁺ -lymphocytes. In contrast, prophylactic treatmentwith Thymalin prevented the latter changes in lymphocyte subpopulations.

Protocol F: 3 years: L-Glu-L-Trp Treatment

An increased incidence of cancer is one sequelae known to occur insubjects exposed to radiation, presumably because of decreased immunesurveillance and elimination of tumor cells. At three yearspost-radiation exposure, preliminary evaluation of patients exposed toradiation at Chernobyl suggested lingering impaired immunity in 20% ofthe subjects as evidenced by reduced numbers of CD2-DR⁺ lymphocytes anddepressed leukocyte enzymes. An attempt to normalize immune parameterswas attempted in 70 of the latter patients exposed 3 years previously to0.2-0.5 Gy of radiation at Chernobyl. Evaluation of these 70 patientsrevealed decreased CD2⁺ -DR⁺ cells (in 100% of the subjects), decreasedsurface IgM⁺ B-lymphocytes (in 49% of the subjects), and nonspecificelevated cytokine production in response to Con-A mitogen in the LMIRassay (in 84% of subjects). In general, most of these patients exhibitedclinical symptoms of secondary immunodeficiency and asthenic syndromeincluding cardiovascular alterations and changes in vital signs. Thesubjects were treated with L-Glu-L-Trp in an attempt to alleviate theimmune defects. The treatment course consisted of daily doses of 100 μgL-Glu-L-Trp administered im for 3-5 days. Sixty three subjects,similarly exposed to radiation in Chernobyl were used as untreatedcontrols. Mean peripheral blood CD2⁺ lymphocyte counts followingL-Glu-L-Trp treatment were restored to within the normal healthy rangeof values (TABLE 6), and a restoration of CD2⁺ counts was observed in85% of the cases. Improvement in the CD2⁺ lymphocyte counts wasaccompanied by improvement in the general condition of the patients anddisappearance of symptoms associated with the asthenic syndrome.Normalization of CD2⁺ lymphocytes did not occur in untreated patients(TABLE 6).

                  TABLE 6                                                         ______________________________________                                        Indices of Cellular Immunity and Innate Immunity in Chernobyl Subject         Receiving Treatment                                                           with L--Glu--L--Trp at 3 Years Post-Radiation Exposure                        Laboratory Test Results                                                       Before          After                                                         Indicia Therapy     Untreated   L--Glu--L--Trp                                ______________________________________                                        Leukocytes,                                                                           5.8 ± 0.3                                                                              5.5 ± 1.0                                                                              5.6 ± 0.4                                  abs                                                                           Lympho- 2.0 ± 0.3                                                                               1.8 ± 0.23                                                                            2.1 ± 0.3                                  cytes, abs                                                                    CD2-DR+,                                                                              15 ± 3*  18.4 ± 2.5                                                                              32 ± 3**                                  CD2-DR+,                                                                               0.30 ± 0.06*                                                                          0.34 ± 0.11                                                                             0.66 ± 0.10**                             abs                                                                           CD3, %  67.7 ± 2.7*                                                                            61 ± 3*   59.2 ± 2.1**                              CD3, abs:                                                                              1.33 ± 0.05*                                                                          1.12 ± 0.18                                                                            1.21 ± 0.15                                CD4, %  36.7 ± 2.6                                                                             38 ± 3   36.2 ± 1.7                                 CD4, abs                                                                              0.72 ± 0.05                                                                            0.70 ± 0.05                                                                            0.74 ± 0.08                                CD8, %  29.7 ± 0.9*                                                                            25.0 ± 2.7                                                                              23.2 ± 2.1**                              CD8, abs                                                                               0.56 ± 0.02*                                                                          0.46 ± 0.06                                                                             0.48 ± 0.07**                             T4/T8    1.24 ± 0.10*                                                                          1.52 ± 0.13                                                                             1.58 ± 0.04**                             LMI, %  140 ± 30*                                                                               107 ± 10**                                                                             75 ± 6**                                  B-Ig+, %                                                                              10.7 ± 0.3                                                                             11.2 ± 0.7                                                                             11.0 ± 0.3                                 B-Ig+, abs                                                                            0.21 ± 0.01                                                                            0.20 ± 0.05                                                                            0.23 ± 0.04                                B-IgM+, %                                                                              3.0 ± 0.3*                                                                            4.4 ± 0.3                                                                               4.1 ± 0.6*                                B-IgM+, abs                                                                            0.062 ± 0.002*                                                                         0.08 ± 0.004                                                                            0.12 ± 0.003**                           B-IgG+, %                                                                             4.7 ± 0.9                                                                              4.6 ± 0.5                                                                              4.8 ± 0.5                                  B-IgG+, abs                                                                           0.059 ± 0.003                                                                           0.08 ± 0.006                                                                           0.09 ± 0.007                              B-IgA+, %                                                                             2.3 ± 0.3                                                                              1.98 ± 0.09                                                                            1.9 ± 0.3                                  B-IgA+, abs                                                                           0.048 ± 0.006                                                                           0.04 ± 0.002                                                                           0.04 ± 0.002                              IgM, g/L                                                                               0.53 ± 0.09*                                                                           1.03 ± 0.13**                                                                          1.06 ± 0.06**                             IgG, g/L                                                                              13.2 ± 1.1                                                                             11.3 ± 1.2                                                                             10.9 ± 1.3                                 IgA, g/L                                                                               0.82 ± 0.25*                                                                          1.1 ± 0.3                                                                               1.2 ± 0.4*                                ______________________________________                                         "a", abs cell concentration presented as 109/L; "b", Post/Pre-Treat Ratio     = posttreatment value/pretreatment value; *statistically significant (p <     0.05) vs. the indices in healthy people; **statistically significant (p <     0.05) vs. the data obtained before treatment; abs cell concentration          presented as 10.sup.9 /L; LMIleukocyte migration inhibition.             

EXAMPLE 3 Radiotherapy-Induced Immunodeficiency Protocol A

Breast Cancer

Thirty six patients with breast cancer were treated with conventionalradiation therapy (i.e., single doses of about 2-3 Gy daily for 10 days;total cumulative patient dose of 20-30 Gy). Following radiotherapy thepatients were treated with L-Glu-L-Trp by injection of daily dosages imof 100 μg. The patients had been previously treated with radiationtherapy (single doses 2 Gy; total dose 20-30 Gy). The results presentedin TABLE 7, show that the L-Glu-L-Trp treatment restored indices ofcellular immunity to normal values.

                                      TABLE 7                                     __________________________________________________________________________    Indices of Cellular Immunity and Innate Resistance in Breast Cancer           Patients Before and After Radiotherapy, and After L--Glu--L--Trp (X ±      m)                                                                                             Before After After                                           Indices          Radiotherapy                                                                         Radiotherapy                                                                        L--Glu--L--Trp                                  __________________________________________________________________________    Lymphocytes (×10.sup.9 /L)                                                               1.61 ± 0.18                                                                       0.79 ± 0.09*                                                                     1.72 ± 0.21**                                Ratio Post-Rad/Pre-Rad Value.sup.a :                                                           (1.0)  (0.5) (1.1)                                           Ratio Post-Treat/Pre-Treat Value:                                                              --     --     (2.18)                                         T-lymphocytes (×10.sup.9 /L)                                                              0.83 ± 0.07+                                                                     0.32 ± 0.03*                                                                     0.92 ± 0.12**                                Ratio Post-Rad/Pre-Rad Value:                                                                  (1.0)  (0.4) (1.1)                                           Ratio Post-Treat/Pre-Treat Value:                                                              --     --     (2.88)                                         "Active" T-lymphocytes (×10.sup.9 /L)                                                    0.49 ± 0.06                                                                       0.19 ± 0.03*                                                                     0.52 ± 0.07**                                Ratio Post-Rad/Pre-Rad Value:                                                                  (1.0)  (0.4) (1.1)                                           Ratio Post-Treat/Pre-Treat Value:                                                              --     --     (2.74)                                         T-helpers (OKT4+)(×10.sup.9 /L)                                                          0.30 ± 0.03                                                                       0.12 ± 0.01*                                                                     0.39 ± 0.04**                                Ratio Post-Rad/Pre-Rad Value:                                                                  (1.0)  (0.4) (1.3)                                           Ratio Post-Treat/Pre-Treat Value:                                                              --     --     (3.25)                                         T-suppressors (OKT8+)(×10.sup.9 /L)                                                      0.28 ± 0.04                                                                       0.16 ± 0.02*                                                                     0.21 ± 0.03                                  Ratio Post-Rad/Pre-Rad Value:                                                                  (1.0)  (0.6) (0.8)                                           Ratio Post-Treat/Pre-Treat Value:                                                              --     --     (1.31)                                         OKT4+/OKT8+      1.07 ± 0.09                                                                       0.75 ± 0.06*                                                                     1.86 ± 0.17**                                DTH.sup.a to tuberculin (mm)                                                                   7.3 ± 0.4                                                                         2.6 ± 0.2*                                                                       8.7 ± 0.6**                                  Ratio Post-Rad/Pre-Rad Value:                                                                  (1.0)  (0.4) (1.2)                                           LMI.sup.b with ConA (%)                                                                        68 ± 4                                                                            96 ± 7*                                                                          71 ± 5**                                     Increase in no. E-RFC in vitro                                                                 1.23 ± 0.15                                                                       1.19 ± 0.13                                                                      1.27 ± 0.14                                  after incubation with L--Glu--L--Trp                                          B-lymphocyte (Ig+)(×10.sup.9 /L)                                                         0.15 ± 0.02                                                                       0.11 ± 0.01                                                                      0.17 ± 0.02                                  Ratio Post-Rad/Pre-Rad Value:                                                                  (1.0)  (0.7) (1.1)                                           Ratio Post-Treat/Pre-Treat Value:                                                              --     --     (1.55)                                         Granulocyte Phagocytic Index                                                                   4.3 ± 0.3                                                                         2.06 ± 0.18*                                                                     3.7 ± 0.2**                                  Granulocyte Cation Proteins                                                                    1.58 ± 0.09                                                                       1.36 ± 0.08*                                                                     1.49 ± 0.12                                  C3-complement (g/L)                                                                            0.75 ± 0.05                                                                       0.66 ± 0.04                                                                      0.68 ± 0.04                                  __________________________________________________________________________     *statistically significant (p < 0.05) vs. the index before radiotherapy;      **statistically significant (p < 0.05) vs. the index after radiotherapy;      a)Delayed Type Hypersensitivity skin reaction to tuberculin (mm diameter)     b)Leukocyte Migration; c)Sensitivity index; "a", Ratio postradiation          value/preradiation value, or the Ratio of the posttreatment (i.e.,            L--Glu--L--Trp) value/pretreatment value.                                

Protocol B Radiation and Chemotherapy

Diffuise alveolar damage and pulmonary disease is reportedly a common,important, and frequently unrecognized problem in patients receivingradiotherapy and chemotherapy (Doran, H. M., et al., Histopathology18(3):211-219 (1991)). Alveolar damage and lung disease in cancerpatients are important targets for therapeutic drug development.

Summary Overview: A total of 246 patients were eventually entered intostudies after receiving radiation and chemotherapy. L-Glu-L-Trp wasadministered to the patients in single 100 μg daily dose for each of 10days during the period of chemotherapy. A control group constituted 158similar patients (following radiation and chemotherapy), and thesepatients were treated in a conventional manner (i.e., chemotherapy andsupport therapy). Patients treated with L-Glu-L-Trp experiencednormalization of immunological indices, and a decreased incidence andseverity of post-operative complications including upper respiratoryinfections, nausea, and inflammations exacerbated by the radiation andchemotherapy, i.e., gastritis, cholecystitis, and the like. Immuneparameters were periodically monitored over the next 4-6 months in thepatients and decreased levels of lymphocytes or their subpopulationswere used as clinical triggers for additional therapeutic interventionwith L-Glu-L-Trp.

Protocol C Tumor Therapy

Thymogen Use in Oncology Practice for the Combined Treatment of Patientswith Thoracic Cavity Tumors: Surgical removal of thoracic tumors wasfollowed in 38 patients by focal radiation therapy and then a course ofL-Glu-L-Trp therapy consisting of 5 daily im injections of 100 μgL-Glu-L-Trp each. Only radiation therapy (and supportive antibiotictherapy) was administered as a control to 56 patients. The clinicalresults that were recorded are summarized in TABLE 8.

                                      TABLE 8                                     __________________________________________________________________________    Clinical Complications in Oncology Patients Treated with                      Focal Radiation Therapy and then L--Glu--L--Trp                                                       Complications.sup.a :                                      No. Radiation                                                                          Therapy                                                                            Immuno-     Cardio-                                        Cancer                                                                             Patients                                                                          Route                                                                              Dose Therapy                                                                            Total                                                                            Septic                                                                            Pulm.                                                                             Mortality                                  __________________________________________________________________________    Esophagus                                                                          22  5-6 Gy/                                                                            20-30 Gy                                                                           5× im                                                                        2  1   1   0                                          Stomach  4 days    100 μg                                                       32            None 10 6   4   0                                          Lung 16  5-6 Gy/                                                                            20-30 Gy                                                                           5× im                                                                        1  0   0   0                                                   4 days    100 μg                                                       24            None 6  3   2   2                                          __________________________________________________________________________     .sup.a Complications, "septic", septic shock; "cardiopulm",                   cardiopulmonary; "mortality", death.                                     

In this trial treatment, L-Glu-L-Trp decreased the total number ofcomplications (Total) as well as the number of cases of septicemia,cardiopulmonary complications, and mortality.

EXAMPLE 4 Respiratory Disease Protocol A: Influeniza Prophylaxis

Summary Overview: A group of 452 persons were treated with daily dosagesof 100 μg of L-Glu-L-Trp administered im on each of 5-10 consecutivedays. A control group consisted of 250 untreated persons. The incidenceof diagnosed respiratory diseases and influenza were recorded for bothgroups over the next four months. The results summarized in TABLES 9 and10 show that the untreated group had a higher incidence of therespiratory disease, illness, hospitalization and disablement than thegroup treated with L-Glu-L-Trp.

                                      TABLE 9                                     __________________________________________________________________________    Prophylactic Treatment with L--Glu--L--Trp: Effect on the Incidence of        Acute Respiratory Diseases and Influenza Illness (mean values)                                            Treatment Group                                                                           Ratio                                 Indices                     L--Glu--L--Trp                                                                        Control                                                                           Control/Tx*                           __________________________________________________________________________    Sickness rate per 100 persons/month                                                                       9.8     30.4                                                                              3.1                                   Pneumonia rate/100 persons/month                                                                          0.20    0.50                                                                              2.5                                   Need for hospitalization, % 30.6    44.9                                                                              1.7                                   Average term of hospitalization, days                                                                     6.2     8.8 1.4                                   Overall incidence of lingering and complicated cases, %                                                   3.9     13.8                                                                              3.5                                   Incidence of lingering and complicated cases for in-patients,                                             9.8     26.2                                                                              2.7                                   Number of cases of temporary disablement per 100 persons/month                                            4.1     7.0 1.7                                   Number of days of temporary disablement per 100 persons/month                                             26.5    57.6                                                                              2.2                                   __________________________________________________________________________     *Ratio = Value Control/Value L--Glu--L--Trp treated.                     

                                      TABLE 10                                    __________________________________________________________________________    Incidence of Acute Respiratory Diseases and Influenza In Recipients of        Prophylactic L--Glu--L--Trp Treatments (mean values) as a Function of         Time After Treatment                                                          Indices       Groups  1st month                                                                          2nd month                                                                          3rd month                                                                          4th month                                __________________________________________________________________________    Sickness rate/100 persons/month                                                             L--Glu--L--Trp                                                                        9.6  11.3 9.4  11.0                                                   Control 28.6 33.4 28.7 30.6                                     Need for hospitalization, %                                                                 L--Glu--L--Trp                                                                        27.0 27.1 28.3 28.3                                                   Control 41.2 50.8 48.8 39.0                                     Average term of                                                                             L--Glu--L--Trp                                                                        6.2  6.2  6.2  7.0                                      hospitalization, days                                                                       Control 9.3  8.4  10.2 11.0                                     Number of cases of temporary                                                                L--Glu--L--Trp                                                                        3.8  7.3  3.4  3.6                                      disablement/100 persons/month                                                               Control 6.9  10.2 7.5  5.6                                      Number of days of temporary                                                                 L--Glu--L--Trp                                                                        24.0 48.6 14.3 26.3                                     disablement/100 persons/month                                                               Control 47.9 82.0 51.4 42.8                                     __________________________________________________________________________

Protocol B: Prophylaxis/Influenza- Athletes in Training:

Summary Overview: The burden of heavy training loads under allconditions of inclement weather places the athlete at an increased riskof respiratory infection. L-Glu-L-Trp was administered to 89 youngsportsmen (athletes). The control group consisted of 54 young students.L-Glu-L-Trp was administered intranasally daily as a single dose of 1μg/kg on each of 3 consecutive days. L-Glu-L-Trp reduced the rate ofupper respiratory infections and illness by 4-fold in the treatmentgroup (i.e., as compared with the controls). In addition, thoseL-Glu-L-Trp treated individuals who did develop an infection had a farless severe course of infection (than in the control group) and theinfections were without complications. Clinical improvement in theL-Glu-L-Trp treatment group was accompanied by the normalization ofimmunological indices.

Trial Population: Students of the Children's and Youth's Sports School(47 boys; 42 girls) were evaluated. The athletic specialties includedtrack and field, rowing, fencing, basketball and swimming. The controlgroup consisted of 30 adolescent boys and 24 girls who did not engage insports. The trial was conducted in the months of January and Februaryand when acute respiratory infections were widespread in studentpopulations.

Therapeutic Protocol: L-Glu-L-Trp was introduced by intranasalinstillation of 8-15 drops of a 0.01% solution daily for 3 days.

Laboratory Indices of Non-Specific Resistance: The followingmeasurements were taken to evaluate the state of non-specific resistanceto infection in the trial and control populations: i) barrier propertiesof the skin and mucous membranes, ii) humoral factors of immunity(bactericidal activity of serum), iii) serum C3 levels, iv) numbers ofneutrophils, their ability to phagocytose yeast, and cellular content ofcationic proteins, v) number of T-lymphocytes (E-RFC), T-suppressor (OKT8⁺) and T-helper cells (OKT 4⁺), vi) blastogenic responses oflymphocytes to PHA and Con-A (% blast cells), vii) number ofB-lymphocytes (EA-RFC), viii) serum concentrations of IgM, IgG, and IgA(radial immunodiffusion), ix) serum levels of immune complexes, and x)levels of lysozyme in saliva. The results of these investigations arepresented in summary form in TABLES 11-13.

                                      TABLE 11                                    __________________________________________________________________________    Blood Hematology Values                                                                      Athletes      Controls                                                              After         After                                      Index          Before                                                                              L--Glu--L--Trp                                                                        Before                                                                              L--Glu--L--Trp                             __________________________________________________________________________    Leukocytes:                                                                   ×10.sup.9 /L:                                                                          5.6 ± 0.2                                                                        6.1 ± 0.2                                                                          6.6 ± 0.9                                                                        5.6 ± 0.4                               % Normal Value:                                                                              (100%)                                                                              (109%)  (100%)                                                                              (97%)                                      Ratio Post-Treat/Pre-Treat Value:                                                            --    (1.09)  --    (0.85)                                     Neutrophils:                                                                  %:             46.5 ± 2.0                                                                       49.0 ± 1.5                                                                         51.5 ± 4.3                                                                       47.5 ± 2.9                              ×10.sup.9 /L:                                                                          2.70 ± 0.09                                                                      3.01 ± 0.2                                                                         3.57 ± 0.8                                                                       2.67 ± 0.3                              % Normal Value:                                                                              (100%)                                                                              (115%)  (100%)                                                                              (75%)                                      Eosinophils:                                                                  %:             4.0 ± 0.6                                                                        4.2 ± 0.6                                                                          3.8 ± 0.6                                                                        4.0 ± 0.8                               ×10.sup.9 /L:                                                                          0.24 ± 0.03                                                                      0.23 ± 0.03                                                                        0.26 ± 0.06                                                                      0.22 ± 0.07                             % Normal Value:                                                                              (100%)                                                                              (96%)   (100%)                                                                              (85%)                                      Monocytes                                                                     %:             8.2 ± 0.4                                                                        9.4 ± 0.5                                                                          9.5 ± 0.9                                                                        10.6 ± 1.2                              ×10.sup.9 /L::                                                                         0.43 ± 0.04                                                                       0.59 ± 0.05†                                                               0.66 ± 0.2                                                                       0.58 ± 0.07                             % Normal Value:                                                                              (100%)                                                                              (137%)  (100%)                                                                              (88%)                                      Lymphocytes:                                                                  %:             38.5 ± 2.0                                                                       34.9 ± 4.7                                                                         34.9 ± 4.7                                                                       37.8 ± 2.9                              ×10.sup.9 /L:                                                                          2.14 ± 0.1                                                                       2.22 ± 0.1                                                                         2.09 ± 0.1                                                                       2.08 ± 0.2                              % Normal Value:                                                                              (100%)                                                                              (104%)  (100%)                                                                              (99%)                                      T-Lymphocytes:                                                                %:             66.4 ± 1.5                                                                       70.8 ± 1.4†                                                                 69.0 ± 1.7†                                                               73.9 ± 1.7                              ×10.sup.9 /L:                                                                          1.43 ± 0.07                                                                      1.55 ± 0.08                                                                        1.45 ± 0.1                                                                       1.56 × 0.2                           % Normal Value:                                                                              (100%)                                                                              (108%)  (100%)                                                                              (108%)                                     T-suppressor (CD8+):                                                          %:             6.3 ± 1.0                                                                        5.9 ± 0.7                                                                          3.6 ± 0.8                                                                        4.8 ± 1.0                               ×10.sup.9 /L:                                                                          0.14 ± 0.02                                                                      0.12 ± 0.02                                                                        0.08 ± 0.03                                                                      0.08 ± 0.05                             % Normal Value:                                                                              (100%)                                                                              (86%)   (100%)                                                                              (100%)                                     T-helper (CD4+):                                                              %:             51.6 ± 1.8                                                                       56.1 ± 1.3†                                                                 49.6 ± 2.9                                                                        65.6 ± 2.0††             ×10.sup.9 /L:                                                                          1.12 ± 0.07                                                                      1.25 ± 0.07                                                                        1.00 ± 0.1                                                                       1.36 ± 0.2                              % Normal Value:                                                                              (100%)                                                                              (112%)  (100%)                                                                              (136%)                                     B-Lymphocytes:                                                                %:             17.7 ± 0.8                                                                        13.6 ± 0.9††                                                        22.4 ± 1.7                                                                       20.1 ± 1.5                              ×10.sup.9 /L:                                                                          0.39 ± 0.02                                                                      0.29 ± 0.02                                                                        0.48 ± 0.07                                                                      0.41 ± 0.05                             % Normal Value:                                                                              (100%)                                                                              (74%)   (100%)                                                                              (85%)                                      IgG (mg/mL)    12.2 ± 0.8                                                                       10.2 ± 0.6                                                                         12.1 ± 0.7                                                                       12.6 ± 0.7                              % Normal Value:                                                                              (100%)                                                                              (84%)   (100%)                                                                              (104%)                                     IgM (mg/mL)     1.1 ± 0.09                                                                       1.1 ± 0.07                                                                         1.3 ± 0.09                                                                        1.0 ± 0.02††            % Normal Value:                                                                              (100%)                                                                              (100%)  (100%)                                                                              (77%)                                      IgA (mg/mL)    1.3 ± 0.6                                                                        1.8 ± 0.1                                                                          1.8 ± 0.2                                                                        1.8 ± 0.2                               % Normal Value:                                                                              (100%)                                                                              (138%)  (100%)                                                                              (100%)                                     C3 (g/L)        0.7 ± 0.02                                                                        0.8 ± 0.02††                                                        1.1 ± 0.06                                                                       0.7 ± 0.03                             Serum Lysozyme (%)                                                                           60.7 ± 1.8                                                                       60.2 ± 1.6                                                                         46.3 ± 1.5                                                                        50.6 ± 1.4††             Serum β-Lysin (%)                                                                       70.3 ± 3.6                                                                        57.6 ± 3.2††                                                        49.8 ± 8.2                                                                       46.8 ± 5.4                              __________________________________________________________________________     †Post-treatment values statistically different than pretreatment       values, p < 0.05;                                                             ††statistically different pre to posttreatment values, i.e.     p < 0.01.                                                                

The investigators concluded that the absolute number of leukocytes inathletes and controls was not significantly different prior totreatment. After treatment with L-Glu-L-Trp, no marked changes wereobserved in PBL, monocytes, lymphocytes, or lymphocyte subpopulations,despite some apparently statistically significant (but small) changes inthe number of monocytes and the percentages of T-helper andB-lymphocytes.

Despite the lack of a significant effect on peripheral blood leukocytes,a statistically significant increase was reported in lysozyme activityin blood (TABLE 11) and saliva (TABLE 12) following treatment withL-Glu-L-Trp. In addition, the phagocytic index of neutrophils (i.e.,number of yeast phagocytosed per cell) was increased (TABLE 13).

                                      TABLE 12                                    __________________________________________________________________________    Nonspecific Resistance to Infection: Salivary Lysozyme, IgA and IgM                      Salivary                                                                            Total   Salivary                                                                             Salivary                                                 Lysozyme                                                                            Salivary                                                                              Secretory                                                                            IgM                                           Group      (%)   IgA (g/L)                                                                             IgA (g/L)                                                                            (g/L)                                         __________________________________________________________________________    Athletes:                                                                     Before:    63.8 ± 2.2                                                                       0.38 ± 0.03                                                                         0.05 ± 0.006                                                                     0.65 ± 0.06                                After L--Glu--L--Trp:                                                                     68.7 ± 1.3†                                                               0.55 ± 0.05††                                                       0.056 ± 0.004                                                                     0.65 ± 0.06                                Controls:                                                                     Before:    54.1 ± 5.5                                                                       0.52 ± 0.1                                                                         0.055 ± 0.009                                                                     0.71 ± 0.06                                After L--Glu--L--Trp:                                                                    63.5 ± 3.0                                                                        0.48 ± 0.1††                                                        0.066 ± 0.009                                                                     0.73 ± 0.09                                __________________________________________________________________________     †statistical significance change from pre to posttreatment at p <      0.05;                                                                         ††p < 0.01                                                 

                                      TABLE 13                                    __________________________________________________________________________    Immune Function                                                                            Athletes      Controls                                           Functional         After         After                                        Indices      Before                                                                              L--Glu--L--Trp                                                                        Before                                                                              L--Glu--L--Trp                               __________________________________________________________________________    Blasts w/PHA (%)                                                                           34.3 ± 1.6                                                                       4.25 ± 2.5††                                                         47.4 ± 6.2                                                                       34.1 ± 3.1                                Blasts w/Con-A (%)                                                                         51.3 ± 3.9                                                                       53.4 ± 3.9                                                                         49.2 ± 5.7                                                                       66.8 ± 4.0††                Phagocytosis (yeast)                                                                       62.4 ± 1.5                                                                       73.3 ± 1.5††                                                         64.8 ± 1.9                                                                       75.0 ± 1.7††                Granulocyte Cationic Proteins                                                               1.4 ± 0.05                                                                      1.1 ± 0.04                                                                          0.95 ± 0.06                                                                     1.22 ± 0.1                                __________________________________________________________________________     †Post-treatment significantly different than the Pretreatment          values, i.e. p < 0.05;                                                        ††posttreatment significantly different that pretreatment       values, p < 0.01                                                         

Clinical Results: In the two month period of the trial, the overallincidence of acute respiratory infection in the students of theChildren's and Youth's Sports School was 63.3% in a total schoolpopulation of 367 adolescents. The rate of infection in the controlgroup of school children not engaged in sports activities was 7.4% (4children). Incidence of infection in the group of athletes treated withL-Glu-L-Trp was 24.7%.

The investigators concluded that under conditions of intense muscularactivity in young athletes there appeared to be a decline in functionalimmune activity as evidenced by decreased LMIR, alteredT-helper/T-suppressor ratios, decreased lymphocyte blast responsivenessto mitogens, decreased circulating B-lymphocytes, and decreased serumimmunoglobulin levels. It was postulated that the presumed immuneimpairments might result from "insufficient maturity of T-cellpopulations." Treatments with L-Glu-L-Trp were seen as favorablyaffecting lysozyme levels, LMIR, IgA levels, and C3 levels (TABLE 11).

Protocol C: Prophylaxis/Respiratory Infections- Polar Explorers:

Summary Overview: L-Glu-L-Trp was administered prophylactically to 27military volunteers with a goal of increasing the subjects' resistanceto high-level solar UV radiation in Northern polar marine climates. Thecontrol group comprised 24 similarly occupied co-workers. L-Glu-L-Trpwas administered intranasally at a dosage of 1 μg/kg daily on each ofthree consecutive days. Prophylactic treatment with L-Glu-L-Trpprevented the occurrence of upper respiratory infections in the treatedgroup which occurred in their co-workers (i.e., the control group).Non-treated subjects exhibited decreases in peripheral blood immuneparameters suggestive of immunosuppression.

Protocol D: Nose, Ear and Throat Infections

Summary Overview: One hundred eighty six subjects (186) with diagnosedacute respiratory disease, including upper airway diseases and viralinfections, were treated with L-Glu-L-Trp. A control group consisted of87 untreated subjects. L-Glu-L-Trp was administered daily im orintranasally at a dosage of 100 μg on each of 3-7 consecutive days.Treatment with L-Glu-L-Trp resulted in a milder course of respiratoryviral infections in the treatment group than in the control group.Clinically evident symptoms of infection were also decreased in thetreated patients, including rhinorrhea, sore throat, fever, muscleaches, headaches, and ear pain. Secondary infectious complications werediminished in the treated individuals, and the length of time thatmedical follow-up was required was also diminished.

Protocol E: Influenza Treatments

One hundred fifty-six patients (156) with influenza were treated with100 μg L-Glu-L-Trp im or 1 μg/kg intranasally on each of 5-10consecutive days. A control group constituted with entry of 82 influenzapatients who remained untreated. Treatments with L-Glu-L-Trp resulted inaccelerated alleviation of symptoms associated with influenza infection,e.g, joint pain, muscle aches, fevers, chills, and upper respiratorysymptoms.

Protocol F: Influenza Symptoms and Sinusitis

Summary Overview: Fifty-one patients (51) were treated daily for 3-10days with either 100 μg L-Glu-L-Trp administered im, or 1 μg/kgadministered intranasally. A control group of 24 similar patientsremained untreated. Treatment with L-Glu-L-Trp resulted in reduced nasalmucous swelling, normalization of breathing, decreased volume of exudatefrom affected sinuses, and improved general condition and immune status.The L-Glu-L-Trp treatment decreased the total duration of patienttreatment by up to 1.7-fold, i.e., as compared with the length oftreatment required for patients in the control group.

Protocol G: Combination Therapy; Influenza Symptoms

Sixty-six (66) hospitalized patients between the ages 18-20 years withconfirmed influenza infections were admitted into the trial. Of thepatients, 12 were determined to be severely infected, and the remaining54 seriously infected and in need of hospitalization. The patients weredivided into two groups, 35 receiving conventional therapy (control),and the remaining 31 received daily injections of 100 μg L-Glu-L-Trp imon each of 3 consecutive days as an adjunct to the conventional ongoingtherapy. The patients treated with L-Glu-L-Trp experienced a 3.5-timeslower incidence of respiratory complications than the patients in thecontrol group. Patients treated with L-Glu-L-Trp also showed a morerapid normalization of immune parameters, including leukocyte migrationinhibition response (LMIR), blastogenic response to PHA and ConA, andlevels of IgA and IgM.

Protocol H: Influenza with Immune Defects

A clinical trial was conducted in a group of 588 patients between theages of 17-21 years all having a confirmed infection with Influenza A,(i.e., confirmation by either hemagglutination-inhibition or thepresence of specific viral antigens). Three hundred fifteen of thepatients (315) were ultimately hospitalized and determined to havesignificant immunological impairment related to their acute viralinfection. Forty-nine patients (49) were treated using a combinationtherapy in which L-Glu-L-Trp was administered as an adjunct to theconventional support therapy. The results of this trial showed thatL-Glu-L-Trp treatment reduced the percentage of patients with secondarycomplications (including pneumonia), and decreased the duration ofpharyngitis, tracheitis, and the time over which therapy needed to beadministered (TABLE 14). Coincident with the improvements in clinicalsymptoms was an observed increase in the percentage of T-helperlymphocytes, and a change in the T-helper/T-suppressor ratio (TABLE 14).

                                      TABLE 14                                    __________________________________________________________________________                   Pharyngitis                                                                              Tracheitis                                                                             Length of Required                         Treatment Regimen                                                                            manifestations                                                                           manifestations                                                                         Therapy                                    __________________________________________________________________________    Combined Therapy:                                                                            3.6 ± 0.3 days                                                                        3.6 ± 0.3 days                                                                      7.1 ± 0.3 days                          Conventional + L--Glu--L--Trp                                                 Conventional Therapy Only                                                                    4.8 ± 0.2 days                                                                        4.0 ± 0.3 days                                                                      8.3 ± 0.4 days                          __________________________________________________________________________                   Secondary (Pneumonia)                                                                    Increase in T-helper                                                                   T-helper/T-suppressor                      Treatment Regimen                                                                            Complications                                                                            Cells Percentage                                                                       Ratio                                      __________________________________________________________________________    Combined Therapy:                                                                            4.0%       17%      2.7                                        Conventional + L--Glu--L--Trp                                                 Conventional Therapy Only                                                                    9.1%        7%      1.8                                        __________________________________________________________________________

Protocol I: Influenza Therapy:

Summary Overview: Forty-eight volunteers (48) were experimentallyinfected with influenza, then examined and treated daily byadministration of L-Glu-L-Trp at a dosage of 100 μg im or 1 μg/kgintranasally over a period of 5-10 days. Thirty-four similarly infectedvolunteers (34) served as untreated controls. Treatment with L-Glu-L-Trpresulted in normalization of fever, reduction in toxic symptoms, andresolution of icterus (jaundice). The hematological and immunologicalindices examined immediately following the period of treatment wererestored to within the normal range.

Protocol J: Influenza:

Summary Overview: L-Glu-L-Trp was administered to 268 volunteers as anadjunct to flu vaccination. The control group consisted of 197 subjectsreceiving flu vaccination but not L-Glu-L-Trp. Flu vaccine was deliveredby air pressure injection and L-Glu-L-Trp was administered daily at adose of 50 μg im on each of 3 consecutive days. L-Glu-L-Trp treatmentsignificantly decreased the incidence of sickness in the vaccinatedsubjects for a period of 12 months compared to controls who receivedflu-vaccination without L-Glu-L-Trp. In the volunteers receivingL-Glu-L-Trp, if they experienced flu, the course of the infection wasnoted to be less severe and the recovery more rapid when compared tocontrols.

Protocol K: Pleuritis:

Summary Overview: Efficacy of L-Glu-L-Trp in combined antibiotic therapywas evaluated in twelve male patients (aged 18 to 45) with suppurativelung diseases that were poorly responsive to antibiotic therapy andaccompanied by diffuse pleuritis, disseminated intravascular coagulation(DIC), pleural effusions, and fever. Laboratory tests revealed elevatedwhite cell counts in peripheral blood, decreased lymphocyte counts(TABLE 17), elevated serum fibrin degradation products (FDP) andabnormally low serum antithrombin III (ATIII) levels (TABLE 15).L-Glu-L-Trp was administered to these patients as a treatment of nearlylast resort, and as part of the ongoing (unsuccessful) regimen of broadspectrum antibiotic therapy: i.e., 200 μg of L-Glu-L-Trp wasadministered im daily over a period of 5-7 days. A dramatic change wasobserved in all 12 patients: namely, fever ablated, the volume ofpleural effusion decreased, incidences of DIC decreased thendisappeared, examination of X-rays showed that infections wereconsolidated, laboratory tests showed decreases in peripheral bloodleukocyte counts, FDP levels dropped, and ATIII levels increased towithin the normal range. In addition, T-cell counts in peripheral bloodincreased and IgG, Igm, and IgA increased (TABLE 17) and C-reactiveprotein levels fell (TABLE 16). No allergic or adverse effects ofL-Glu-L-Trp treatment were observed. The results of the laboratory testsconducted in this trial are summarized in TABLES 15-17.

                  TABLE 15                                                        ______________________________________                                        Hematology Values                                                                               Patients   After                                                              Before     L--Glu--L--Trp                                   Indicia           Therapy    Therapy                                          ______________________________________                                        Clotting time (citrated whole blood-                                                            116.2 ± 11.6                                                                          146.5 ± 28.2                                  recalcified)                                                                  Kaolin-Cerphalin clotting time                                                                  54.1 ± 5.7                                                                            61.7 ± 6.1                                    Fibrinogen Concentration (g/L)                                                                   6.9 ± 1.6                                                                            4.7 ± 1.2                                     FDP (μg/mL)      156 ± 24.7                                                                          21.7 ± 0.8†                            ATIII (% of normal)                                                                              69.5 ± 21.2                                                                          98.3 ± 15.3                                   Fibrinolytic Activity (min.)                                                                    265.2 ± 54.0                                                                          181.2 ± 52.2                                  ______________________________________                                         †Statistically different than the normal control values, i.e., p <     0.05.                                                                    

                  TABLE 16                                                        ______________________________________                                        Blood Acute-Phase Protein Measurements                                                       Patients   After                                                              Before     L--Glu--L--Trp                                      Indicia        Therapy    Therapy                                             ______________________________________                                        C3 complement (mcg/mL)                                                                         735 ± 46.8                                                                          918.9 ± 85.9†                             Prealbumin (mcg/mL)                                                                          267.5 ± 42.5                                                                          273.3 ± 40.7                                     Ceruloplasmin (μg/mL)                                                                       321 ± 34.8                                                                           295 ± 22.8                                      Orosomucoid (μg/mL)                                                                       1368.3 ± 181.7                                                                        1146.7 ± 147                                     α2-macroglobulin (μg/mL)                                                             1.8 ± 0.2                                                                            2.4 ± 0.2                                        α1-anti-trypsin (μg/mL)                                                             185.5 ± 15.6                                                                          96.0 ± 3.5                                       C-reactive protein (μg/mL)                                                                36.8 ± 2.6                                                                             7.4 ± 2.5†                               Transferrin (μg/mL)                                                                        2.8 ± 0.8                                                                            3.2 ± 0.4                                        ______________________________________                                         †Statistically different than the normal control values, i.e., p <     0.05.                                                                    

                  TABLE 17                                                        ______________________________________                                        Laboratory Indicia of Disease Activity in Patients with                       Lung and Pleural Disease                                                                               After                                                               Before    L--Glu--L--Trp                                       Indicia        Treatment Treatment                                            ______________________________________                                        T-Lymphocytes:                                                                ×10.sup.9 /L:                                                                          0.46 ± 0.3                                                                           0.74 ± 0.08†                               % Normal Value:                                                                              (52%)     (83%)                                                B-Lymphocytes:                                                                ×10.sup.9 /L:                                                                           0.35 ± 0.01                                                                         0.46 ± 0.02†                               % Normal Value:                                                                              (76%)     (100%)                                               IgG (mg/mL)    153.2 ± 12.6                                                                         186.8 ± 31.8                                      % Normal Value:                                                                              (114%)    (139%)                                               IgM (mg/mL)    140.8 ± 32.2                                                                         131.2 ± 38.5                                      % Normal Value:                                                                              (111%)    (103%)                                               IgA (mg/mL)    153.4 ± 25.6                                                                         1595. ± 12.3                                      % Normal Value:                                                                              (156%)    (162%)                                               ______________________________________                                         †Statistically different than the normal control values, i.e., p <     0.05.                                                                    

Protocol L: Pneumonia

Eight children with progressive destructive pneumonia were treated withL-Glu-L-Trp in combination with conventional antibiotic therapy. Allpatients presented at the hospital with marked manifestations ofsystemic toxicity, in serious condition, and all with respiratoryimpairment. In all cases there was also diagnosis of secondary immunedeficiency and thrombohemorrhagic syndrome. L-Glu-L-Trp was administereddaily at a dosage of 100 μg im daily for 5 days. Laboratory tests wereconducted to compare the pre- and post-treatment values with valuesrecorded for normal healthy controls. A control population of childrenwith pneumonia was treated with antibiotic therapy alone. The results ofthe laboratory analyses are presented in TABLES 18-20.

                                      TABLE 18                                    __________________________________________________________________________    Hematology Values                                                                               Normal                                                                              Patients                                                                             After                                                            Healthy                                                                             Before L--Glu--L--Trp                                 Index             Control                                                                             Therapy                                                                              Combination Therapy                            __________________________________________________________________________    Whole blood clotting time (sec.)                                                                472 ± 18.4                                                                       360 ± 23.5                                                                        465 ± 16.2                                  Clotting time (citrated whole blood-                                                            142 ± 8.9                                                                        133.4 ± 4.3                                                                       129 ± 2.3                                   recalcified)                                                                  Kaolin-Cephalin clotting time                                                                   62 ± 4.1                                                                         51 ± 1.7                                                                          58 ± 1.2                                    Prothrombin time  22 ± 0.5                                                                         26 ± 0.2                                                                          22 ± 0.1                                    Thrombotest       41 ± 2.3                                                                         33 ± 1.2                                                                          33 ± 1.2                                    Fibrinogen Concentration (g/L)                                                                  4.51 ± 0.65                                                                      5.1 ± 0.2                                                                         4.2 ± 0.4                                   FDP (μg/mL)    128 ± 11.2                                                                       178 ± 13.5                                                                        96 ± 6.7                                    ATIII (% of normal)                                                                             83 ± 4.4                                                                         78 ± 1.8                                                                          85 ± 2.1                                    Fibrinolytic Activity (min.)                                                                    204 ± 16.3                                                                       245 ± 13.7                                                                        186 ± 11.5                                  Hagemann Factor Dependent Fibrinolysis                                                           79 ± 10.7                                                                       118 ± 18.4                                                                        72 ± 6.8                                    __________________________________________________________________________

Unfortunately no p-values were calculated for the data presented inTABLES 18-20 obtained in this study in the U.S.S.R. However, thefindings presented in TABLE 18 suggested underlying thrombosis in thispatient population with elevated levels of fibrin degradation products(FDP) and decreased levels of ATIII. L-Glu-L-Trp treatment apparentlydecreased the levels of fibrin degradation products; and increased serumATIII levels. (Similar effects of L-Glu-L-Trp on FDP and ATIII wereobserved in studies of patients with disseminated intravascularcoagulation resulting from pleuritis, Example 4, Protocol E, above.)

                  TABLE 19                                                        ______________________________________                                        Laboratory Indicia of Disease Activity in Children with                       Bacterial Pneumonia                                                                                           After                                                   Normal                Combination                                             Healthy    Before     L--Glu--L--Trp                                Marker    Control    Therapy    Therapy                                       ______________________________________                                        Leukocytes                                                                    ×10.sup.9 /L:                                                                     11.2 ± 0.1                                                                            13.3 ± 0.1                                                                            11.0 ± 0.01                                % Normal Value:                                                                         (100%)     (119%)     (98%)                                         Lymphocytes                                                                   ×10.sup.9 /L:                                                                      0.31 ± 0.03                                                                          0.335 ± 0.002                                                                         0.36 ± 0.01                                % Normal Value:                                                                         (100%)     (108%)     (116%)                                        T-Lymphocytes:                                                                ×10.sup.9 /L::                                                                    0.681 ± 0.09                                                                           0.9 ± 0.09                                                                            1.1 ± 0.01                                % Normal Value::                                                                        (100%)     (132%)     (167%)                                        B-Lymphocytes:                                                                ×10.sup.9 /L:                                                                     0.482 ± 0.07                                                                          0.70 ± 0.05                                                                           0.84 ± 0.06                                % Normal Value:                                                                         (100%)     (145%)     (175%)                                        IgG (mg/mL)                                                                              142 ± 9.8                                                                             133 ± 6.6                                                                             138 ± 5.6                                 % Normal Value:                                                                         (100%)     (94%)      (97%)                                         IgM (mg/mL)                                                                               171 ± 13.6                                                                           160 ± 6.9                                                                             153 ± 6.9                                 % Normal Value:                                                                         (100%)     (94%)      (89%)                                         IgA (mg/mL)                                                                               41 ± 5.2                                                                             60 ± 5.9                                                                              67 ± 4.8                                  % Normal Value:                                                                         (100%)     (146%)     (163%)                                        ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        Clinical Indicia of Disease Activity in Children with                         Bacterial Pneumonia                                                                             Conventional                                                                             L--Glu--L--Trp                                                     Antibiotic Combination                                      Clinical Index    Therapy    Therapy                                          ______________________________________                                        Duration Systemic Toxicity (days)                                                               10.7 ± 1.6                                                                             8.2 ± 0.9                                    Patient Condition Stabilized (days)                                                             10.6 ± 2.0                                                                             6.8 ± 0.7                                    Normalization of Body Temperature                                                               20.5 ± 2.4                                                                            13.9 ± 1.7                                    (days)                                                                        Favorable Resolution by X-ray (days)                                                            21.9 ± 3.1                                                                            19.3 ± 2.5                                    Hemoglobin (g/L)  106.0 ± 2.7                                                                           115.0 ± 1.6                                   Erythrocyte Sedimentation Rate (mm)                                                             34.8 ± 4.8                                                                            26.2 ± 3.2                                    Antibiotics Required (days)                                                                     25.9 ± 2.0                                                                            21.4 ± 1.4                                    Hospital Bed-Days 42.3 ± 2.1                                                                            41.7 ± 1.5                                    Mortality %       1.2        0                                                ______________________________________                                    

Clinical Effects: L-Glu-L-Trp treated patients showed a normalization ofbody temperature more rapidly than control patients receivingconventional antibiotic therapy, and in patients with intrapulmonaryinfection, the infection resolved more rapidly than in controls, asevidenced by changes in lung opacity on chest X-ray.

EXAMPLE 5 Acquired Immunodeficiency Syndrome

In separate studies, a total of 21 HIV-infected individuals werestudied, including fill-blown syndrome, prodromal, and pre-AIDSafflicted individuals who were treated with Thympentin. Thympentin andTPI are thymic gland peptide extracts previously disclosed in thescientific literature. Previous comparative studies reveal L-Glu-L-Trpto be more effective than TPI in restoring normal immunologic indices,T-cell functional activity, and T4/T8 ratios.

Method of Administration: Sterile saline containing the sodium salt ofL-Glu-L-Trp can be administered either im, or by intralymphatic, orintranasal routes each day for 5-10 consecutive days, and the treatmentsare repeated every 30 days.

As reported in Examples 2 and 3, above, immunosuppressed individuals whohave sustained radiation injuries and were treated with L-Glu-L-Trpexhibited excellent restoration of immunological indices. Therefore,L-Glu-L-Trp will benefit HIV infected individuals, and AIDS patients, bytreating symptoms and complications resulting from the acute HIV viralinfection and thereby reducing the need to use other medications withpotentially toxic side effects.

As shown in the Examples below, L-Glu-L-Trp treatments stimulated innateimmunity and anti-bacterial cellular immunity in experimental animals.Stimulation of the latter immune mechanisms in HIV infected (or AIDS)patients will result in a reduction in the incidence, duration, orseverity of opportunistic infections including respiratory infections.

EXAMPLE 6 Dermatologic Diseases

Patients were entered into a large ongoing dermatology clinical trialprotocol, the results of which are summarized in Protocols A-F, below.

Protocol A: Chronic Staphylococcal Pyoderma:

Summary Overview: The total clinical trial population eventually underexamination consisted of 159 patients with pyoderma, includingfurunculitis, cellulitis, and folliculitis. Trial Protocol A1, below,details some of the results obtained in this trial. Medications wereadministered either im or intranasally for 5 consecutive days.Peripheral blood immune indices were decreased prior to treatment andfollowing treatment with L-Glu-L-Trp were restored to within the normalrange. Treatments with L-Glu-L-Trp also resulted in a disappearance ofskin manifestations and resolution of pyoderma in the treated patients.Clinical improvement was correlated with return of the immune parametersto within the normal range of values. In certain patients, L-Glu-L-Trpwas also topically applied as a sterile saline solution of medicationfor a period of 5 to 10 days at a dosage of 1 μg/kg body weight.

Protocol A1: Chronic Staphylococcal Pyoderma:

Patient Population: Efficacy of L-Glu-L-Trp in combined antibiotictherapy was evaluated in 59 patients (aged 18 to 56; 32 men and 27women) with chronic pyoderma non-responsive to antibiotic therapy. Thepatients were divided into Group 1 (n=36; L-Glu-L-Trp combinationtherapy) and Group 2 (n=23; control), as described below. Diagnosis, atthe time of clinical presentation, and the demographics of the patientpopulation are summarized in TABLE 21. The length of illness for thepatients in the trial ranged from 5 months to 16 years.

                                      TABLE 21                                    __________________________________________________________________________    Patient Population                                                                           Total                                                                             Demographics                                               Patient        No. Male Ages:  Female Ages:                                   Diagnosis      Patients                                                                          18-30                                                                             31-43                                                                             44-56                                                                             18-30                                                                             31-43                                                                             44-56                                  __________________________________________________________________________    Chronic relapsing osteofolliculitis                                                          3   1   --  --  1   1   --                                     Volar eruptions                                                                              1   --  1   --  --  --  --                                     Papular-pustular acne                                                                        19  12  --  --  6   1   --                                     Chronic relapsing folliculitis gravis                                                        1   --  --  --  --  1   --                                     Chronic furunculitis                                                                         11  1   2   1   4   1   2                                      Abscess and indurative acne                                                                  14  7   --  --  6   1   --                                     Chronic relapsing hydradenitis                                                               1   1   --  --  --  --  --                                     Chronic abscess pyoderma                                                                     7   4   2   --  --  --  1                                      Chronic ulcerative pyoderma                                                                  2   --  --  --  --  2   --                                     Totals:        59  26  5   1   17  7   3                                      __________________________________________________________________________

Group 1 consisted of 36 patients (18-56 years of age; 17 men and 19women) who had been previously repeatedly treated with antibioticswithout significant clinical effect. Staphylococci cultured from 14patients showed broad spectrum resistance to 10 of 15 antibioticstested. The length of illness was from 6 months to 16 years. Twenty-fourof the patients (24) had a clinical history that included recurrence ofone or more diseases, e.g., chronic tonsillitis, maxillary sinusitis,dental granuloma, periodontitis, pancreatitis, gastritis, gastriculcers, chronic bronchitis, chronic prostatitis, otitis, etc.Staphylococci were isolated from dermal foci of infection in all 30patients. The latter isolates were resistant to several antibiotics andin 14 patients, the staphylococci were multiple drug resistant, (i.e.,resistant to 10 of the 15 antibiotics tested).

Group 2 (conventional treatment control ) consisted of 23 patients withsimilar demographics and pyoderma diseases.

Therapeutic Protocols: The patients in Group 1 were treated in acombination, two-stage therapy using antibiotics and L-Glu-L-Trp.Patients in Group 2 received only antibiotics and other palliativetherapies.

Stage 1: In the first stage of the protocol, only L-Glu-L-Trp wasadministered in the hope of increasing host immune responsiveness andantibiotic sensitivity of the bacteria. At the conclusion of stage 1,slaphylococci were isolated from the patients and tested for drugresistance. Based on the results of the antibiotic sensitivity testing,an antibiotic was selected for use in Stage 2, below.

Stage 2: In the second stage, both L-Glu-L-Trp and the selectedantibiotic were administered in combination, and in certain cases astaphylococcal "autovaccine" was prepared and used to immunize thepatients against their individual staphylococcal strain. The decision onthe type of second stage therapy was made on a patient-by-patient basisafter evaluating the effects of first stage therapy on B-lymphocytes,neutrophils, and IgM levels in circulation. If these indicators weresignificantly elevated then only combination therapy with L-Glu-L-Trpand antibiotic was administered in the second stage, however, if theindicators were not elevated, then autovaccine was administered inaddition to the antibiotic and L-Glu-L-Trp therapy, i.e., in an attemptto boost an immune response to the bacteria.

Treatment Routes: L-Glu-L-Trp treatment was administered by twodifferent routes: namely, im (Group 1A) and intranasally (Group 1B).Group 1A consisted of 17 patients (i.e., 17 of the 36 patients inGroup 1) who were treated with L-Glu-L-Trp by intramuscular injection of100,ug daily for 5 days. Group 1B, the remaining 19 patients in Group 1,received L-Glu-L-Trp by intranasal instillation, daily for 5 days, of 1mL of a 0.01% solution. (In pharnacokinetic studies of experimentalanimals, administration of labeled L-Glu-L-Trp by intranasalinstillation resulted in rapid, i.e., minutes, appearance of label inperipheral blood.) Results obtained with Group 1A and Group 1B were notstatistically different, so the data were combined for subsequentanalysis and the data obtained with Groups 1A and 1B are presentedtogether in tabular form below.

Antibiotics were administered according to established dosages androutes: i.e., penicillin, ampicillin, oxacillin, tetracycline,erythromycin, garamycin; administered by intramuscular route.Antibiotics were chosen according to the antibiotic sensitivity of theStaphylococci isolated from the respective patients. (Antibioticsensitivity was determined by testing staphylococcal isolates for theirsensitivity in an antibiotic sensitivity doubling-dilution assay withthe aid of the semi-automatic MIC-2000 system, Dynatech, USA).

Laboratory Test Results: Materials and methods used in these studiesappear in the text immediately following EXAMPLE 34, below. Laboratoryparameters of disease activity were monitored including, morning bodytemperature, leukocyte and differential blood cell counts, and immuneparameters (TABLE 22). The mean values (+/-S.D.) recorded in Group 1Aand 1B patient samples at the conclusion of Stage 2 of therapy aresummarized in TABLE 22.

                                      TABLE 22                                    __________________________________________________________________________    Laboratory Indicia of Disease Activity in Patients with Chronic Pyoderma                         Conventional                                                           Normal Treatment                                                                              L--Glu--L--Trp.sup.a                                          Healthy                                                                              Control  Treatment                                         Marker      Value  (n = 23) (n = 36)                                          __________________________________________________________________________    Leukocytes                                                                    (×10.sup.9 /L):                                                                     6.71 ± 0.17                                                                        7.89 ± 0.36††                                                        7.75 ± 0.33                                    % Normal Value:                                                                           (100%) (118%)   (115%)                                            Lymphocytes:                                                                  %:           28 ± 0.6                                                                         29.4 ± 1.3                                                                          30.8 ± 1.4                                     ×10.sup.9 /L:                                                                       2.01 ± 0.09                                                                       2.26 ± 0.12                                                                         2.41 ± 0.14                                    % Normal Value:                                                                           (100%) (112%)   (120%)                                            T-Lymphocytes:                                                                %:          61.4 ± 1.6                                                                        62.8 ± 1.9                                                                          69.3 ± 1.9*                                    ×10.sup.9 /L::                                                                      1.70 ± 0.12                                                                        1.38 ± 0.08†                                                                 1.68 ± 0.12*                                  % Normal Value::                                                                          (100%) (81%)    (99%)                                             T-helper: (OKT4+)                                                             %:          35.3 ± 2.7                                                                        13.7 ± 1.6†                                                                  20.3 ± 2.9                                     ×10.sup.9 /L:                                                                       0.65 ± 0.05                                                                        0.30 ± 0.03†                                                                 0.46 ± 0.05*                                  % Normal Value::                                                                          (100%) (46%)    (71%)                                             T-suppressor: (OKT8+)                                                         %:          21.3 ± 0.9                                                                        17.9 ± 1.4                                                                          17.1 ± 1.9                                     ×10.sup.9 /L:                                                                       0.41 ± 0.03                                                                       0.37 ± 0.04                                                                         0.39 ± 0.04                                    % Normal Value:                                                                           (100%) (90%)    (95%)                                             T4.sup.+ /T8.sup.+  Ratio:                                                                1.64 ± 0.12                                                                        0.81 ± 0.17†                                                                 1.32 ± 0.11*                                  B-Lymphoctyes:                                                                %:          18.1 ± 1.4                                                                        13.5 ± 1.4                                                                          12.1 ± 1.5                                     ×10.sup.9 /L:                                                                       0.49 ± 0.04                                                                        0.30 ± 0.04††                                                        0.28 ± 0.04                                    % Normal Value:                                                                           (100%) (61%)    (57%)                                             Surface Ig+ B-Lym:                                                            %:          13.8 ± 1.2                                                                        16.1 ± 2.2                                                                          16.9 ± 1.7                                     ×10.sup.9 /L:                                                                       0.29 ± 0.02                                                                       0.40 ± 0.07                                                                         0.34 ± 0.05                                    % Normal Value:                                                                           (100%) (138%)   (117%)                                            IgG (g/L)   11.5 ± 0.50                                                                        14.1 ± 0.49††                                                        15.5 ± 0.73                                    % Normal Value:                                                                           (100%) (123%)   (135%)                                            IgM (g/L)   1.15 ± 0.06                                                                       1.38 ± 0.17                                                                         1.25 ± 0.12                                    % Normal Value:                                                                           (100%) (120%)   (109%)                                            IgA (g/L)   1.90 ± 0.08                                                                        2.38 ± 0.14††                                                        2.40 ± 0.18                                    % Normal Value:                                                                           (100%) (125%)   (126%)                                            C3 (g/L)    0.84 ± 0.02                                                                        0.76 ± 0.01††                                                        0.77 ± 0.02                                    % Normal Value:                                                                           (100%) (90%)    (92%)                                             Imm. Cmplx. (Units)                                                                       44.0 ± 1.6                                                                        39.6 ± 3.6                                                                          45.9 ± 4.9                                     NK Activity (% cytotox.)                                                                  45.07 ± 2.82                                                                       29.8 ± 3.35†                                                                45.18 ± 2.55*                                  Monocytes:                                                                    %:           7.0 ± 0.28                                                                       6.14 ± 1.4                                                                          5.65 ± 0.40                                    ×10.sup.9 /L:                                                                       0.55 ± 0.03                                                                       0.48 ± 0.03                                                                         0.43 ± 0.04                                    % Normal Value:                                                                           (100%) (87%)    (78%)                                             Neutrophils:                                                                  %:          63.0 ± 1.1                                                                        61.1 ± 1.5                                                                          59.8 ± 1.5                                     10.sup.9 /L 4.41 ± 0.18                                                                       4.98 ± 0.29                                                                         4.54 ± 0.25                                    % Normal Value::                                                                          (100%) (113%)   (103%)                                            Phagocytic Cells                                                              ×10.sup.9 /L:                                                                       2.65 ± 0.06                                                                        2.19 ± 0.10†                                                                2.36 ± 0.27                                    % Normal Value:                                                                           (100%) (83%)    (89%)                                             Phag. Index 4.92 ± 0.05                                                                        4.51 ± 0.58†                                                                 5.31 ± 0.20**                                 Erythrocytes ×10.sup.12 /L                                                          --     5.4 ± 0.7                                                                           5.2 ± 0.6                                      Erythrocyte Sed. Rate (mm)                                                                --     13.2 ± 1.3                                                                          11.2 ± 1.4                                     __________________________________________________________________________     a. L--Glu--L--Trp therapy = 1st stage therapy with L--Glu--L--Trp only        described below under "Clinical Results"; b. HSA, hemolytic Staphylococcu     aureus antigen; †, Significant different between healthy/normal an     pretreatment values at the p < 0.05 level; ††, p < 0.01         level; or †††, p < 0.001 level; *, Significant           difference between patient pre and posttreatment values at the p < 0.05       level; **, p < 0.01 level; or, ***, p < 0.001 level.                     

T-Lymphocytes: The results presented in TABLE 22 show i) that patientswith chronic pyoderma have decreased peripheral blood T-lymphocytes, T4⁺-lymphocytes, and NK activity similar to those existent in certainimmunodeficiency states; and, ii) that treatment with L-Glu-L-Trpstimulated an increase in peripheral T-lymphocytes, T4⁺ lymphocytes, theratio of T4⁺ /T8⁺ lymphocytes, and NK activity in peripheral bloodto >70% of values recorded in peripheral leukocytes of normal healthyvolunteers.- Functional activity of peripheral T-lymphocytes, asmeasured by blastogenesis in response to stimulation with Con-A and PHAmitogens and HSA (hemolytic staphylococcal allergen) antigen, was alsoapparently depressed in chronic pyoderma patients and treatment withL-Glu-L-Trp increased blastogenesis to greater than (or near equal to)the levels produced by healthy volunteers (TABLE 23).

                  TABLE 23                                                        ______________________________________                                        Lymphocyte Blastogenic Activity:                                                          Normal    Patients  After                                         Blast Transformation                                                                      Healthy   Before    L--Glu--L--Trp                                (Antigen)   Values    Therapy   Therapy                                       ______________________________________                                        PHA (%)     35.9 ± 2.6                                                                           50.2 ± 2.7                                                                           40.2 ± 3.9                                 Con-A (%)   49.4 ± 3.3                                                                           76.3 ± 4.9                                                                           58.3 ± 3.9                                 HSA (%)     95.1 ± 5.7                                                                           114.5 ± 7.6                                                                          84.9 ± 5.5                                 ______________________________________                                         (No mean values were significantly different than the Pretreatment values     i.e. p > 0.05)                                                           

Multivariant analysis of the immune values and clinical results observedin these studies suggested a direct correlation between the length ofstaphylococcal infection and the blood levels and immune functions of T-lymphocytes. The results of this trial suggest that immunity mediatedby T-lymphocytes may not express itself in chronic staphylococcal skindisease until >2.5-4 years after diagnosis of the infection, despite (orbecause of) treatment with conventional antibiotics and corticosteroidointments. In this patient population, antibiotic resistance wasapparently correlated with the development of a possible T-lymphocytedeficiency. Treating the patients' immunodeficiency with L-Glu-L-Trp wasapparently correlated with a decrease in antibiotic resistance of thestaphylococci isolated from the patients.

B-Lymphocytes: The results presented in TABLE 22, show decreasedabsolute B-lymphocyte levels and surface Ig⁺ lymphocytes in the trialpopulation, despite increased numbers of surface IgG⁺ lymphocytes andincreased serum levels of IgG and IgA in the peripheral blood samples.Following Stage 2 of L-Glu-L-Trp treatment, total B-lymphocyte countsdid not change significantly, but the numbers of surface IgG⁺lymphocytes decreased, i.e., from 0.37±0.075×10⁹ /L to 0.19±0.023×10⁹ /L(p<0.05) as compared to normal levels of 0.08±0.008×10⁹ /L.

Phagocytic Activity: The results presented in TABLE 22 show an increasein the number of Staphylococci ingested by each peripheral bloodphagocytic cell (i.e., phagocytic index), a favorable fuinctionalcharacteristic commonly observed with "activated" macrophages andneutrophils.

In vitro studies: In a separate in vitro study, lymphocytes from 26patients of Group 1, above, were incubated in tissue culture mediumcontaining 0.01% L-Glu-L-Trp (i.e., 100 μg/mL). An increase in thepercentage of E-RFC was observed from 64.2±2.27% to 73.3±2.26% (p<0.01),OKT4⁺ lymphocytes increased from 13.4±1.4% to 18.2±1.6% (p<0.05), butOKT8⁺ lymphocytes did not change significantly, i.e., 17.2±1.7% to17.9±1.4% (p>0.05). (Similar data showing increased OKT4 expression onT-lymphocytes treated in vitro with L-Glu-L-Trp is presented in otherExamples, below.)

Clinical Results:

Stage 1: Clinical results recorded during the first stage of therapy(L-Glu-L-Trp only), included a short-lived aggravation of the dermaldiseases in 20 of the 36 patients which occurred after the second day ofL-Glu-L-Trp therapy. The aggravation was manifest as an "intensificationof hyperemia and dermal infiltration around infected foci and anincrease in the amount of purulent excretion." During continuedadministration of L-Glu-L-Trp the "phenomenon subsided" and"regenerative processes were strengthened". In the cutaneous lesionsacceleration of scar tissue formation and re-epithelialization wereobserved. Bacterial isolates were tested for antibiotic sensitivity, andincreased antibiotic sensitivity was recorded for isolates from certainpatients. At the time of the trial it was thought possible thatL-Glu-L-Trp treatment induced synthesis and release ofanti-staphylococcal factor (ASF), a T-cell cytokine that reportedlyincreases the antibiotic sensitivity of antibiotic-resistantStaphylococci.

Staphylococci were isolated from patients before and after treatment inStage 1 with L-Glu-L-Trp in order to determine whether a favorablereversal of T-lymphocyte immunodeficiency might lead to a change in theantibiotic sensitivity. The isolated bacterial colonies following Stage1 were typed as follows: namely, in 16 patients S. aureus, in 34, S.epidermidis, in 2 saprophytic Staphylococci. In all cases the same typesof Staphylococci were isolated following treatment with L-Glu-L-Trp, butin 4 patients the predominant Staphylococci in the isolates changed fromS. aureus to S. epidermidis (n=2), and saprophytic Staphylococci (n=2).The results of testing patient isolates for antibiotic sensitivity aresummarized in TABLE 24.

                  TABLE 24                                                        ______________________________________                                        Antibiotic Sensitivity of Patient Isolates                                                 Mean Sensitivity (μg/mL)                                                               After                                                               Before    Stage 1                                              Antibiotic     Treatment Treatment                                            ______________________________________                                        Penicillin     9.45 ± 0.28                                                                          2.52 ± 0.70                                       Bicillin-3     9.24 ± 0.53                                                                          2.42 ± 1.02                                       Oxacillin      5.11 ± 0.63                                                                          1.56 ± 0.45                                       Ampicillin     8.10 ± 0.49                                                                          2.64 ± 0.60                                       Levomycetin    9.17 ± 0.36                                                                          4.58 ± 0.67                                       Streptomycin   6.71 ± 0.59                                                                          3.39 ± 0.66                                       Monomycin      6.34 ± 0.55                                                                          2.01 ± 0.46                                       Kanamycin      6.32 ± 0.58                                                                          2.09 ± 0.54                                       gentamycin     5.47 ± 0.92                                                                          1.32 ± 0.49                                       Cisomycin      3.46 ± 0.90                                                                          1.59 ± 0.58                                       Ceporin        5.69 ± 0.86                                                                          1.82 ± 0.67                                       Cephamezine    3.52 ± 0.94                                                                          1.33 ± 0.53                                       Keflin         4.49 ± 0.93                                                                          1.86 ± 0.69                                       Kefzol         4.61 ± 0.87                                                                          1.86 ± 0.93                                       ______________________________________                                    

The results presented in TABLE 24 show that the Staphylococci isolatedfrom patients prior to therapy were relatively resistant to penicillinantibiotics, however the isolates were relatively sensitive in vitro tocephalosporins (i.e., Ceporin, Cephan, Keflin, and Kefzol), and certainaminoglycoside antibiotics (i.e., Cisomycin). Following treatment withL-Glu-L-Trp the Staphylococci isolated from the patients showedincreased antibiotic-sensitivity to one, a few, or all antibiotics(noteworthy exceptions being penicillin, bicillin-3, and levomycetin).In certain cases more than a 100-fold increase in antibiotic sensitivitywas observed. Thus, it may be concluded that while L-Glu-L-Trp did notcompletely eliminate penicillin resistance, the phenotype of multipledrug resistance (i.e., to antibiotics other than penicillin) was alteredfollowing treatment.

Stage 2: Clinical results recorded during the second stage of therapy(i.e., with L-Glu-L-Trp and an antibiotic, pyrogenal, or autovaccine,above), are summarized in TABLE 25. The clinical findings includedabsence of suppuration from dermal infections, and absence of neweruptions of infected foci from the skin as well as completecicatrization of ulcers and resolution of deep infiltrates. Peripheralblood leukocyte counts also returned to normal values by the end of thetherapy and humoral immune activity also appeared to be increased asmeasured by the absolute levels of serum IgM.

                                      TABLE 25                                    __________________________________________________________________________    Clinical Results: Following Stage 2 of Treatment:                                            Number of Patients with Clinical Result:                                           Significantly                                                                            No                                             Patient Diagnosis                                                                            Recovered                                                                          Improved                                                                            Improved                                                                           Effect                                                                            Worse                                      __________________________________________________________________________    Chronic relapsing osteofolliculitis                                                          3    --    --   --  --                                         Chronic relapsing folliculitis gravis                                                        1    --    --   --  --                                         Papular-pustular acne                                                                        4    3     --   --  --                                         Abscess and indurative acne                                                                  4    1     1    --  --                                         Chronic furunculitis                                                                         10   --    --   --  --                                         Chronic abscess pyoderma                                                                     5    2     --   --  --                                         Chronic ulcerative pyoderma                                                                  --   2     --   --  --                                         Totals:        27   8     1    0   0                                          __________________________________________________________________________

Long term follow-up: Combination therapy with L-Glu-L-Trp andantibiotics resulted in clinical recovery in 27 patients with chronicpyoderma who previously had recurrently failed conventionalbroad-spectrum antibiotic therapy. In addition, considerable improvementwas noted in 8 patients and improvement was observed in 1 case. In the24 latter patients, recovery was stable over a six month period, and inthe remaining 3 patients (of the trial) the recurrences of infectionwere much more mild than those previously documented in the patients'medical histories. Clinical recovery was attained in all patients withchronic recurring osteofolliculitis and folliculitis as well as thosewith chronic furunculosis. Of 7 patients with papulous pustular acne,clinical recovery was achieved in 4. Of 10 patients with abscessing andindurative acne, clinical recovery was achieved in 6. Of 7 patients withchronic abscessing pyoderma, complete clinical recovery with resolutionof infiltrates and cicatrization of ulcers was achieved in 5. During6-12 months follow-up, relapses were observed in 2 patients with acnevulgaris and 1 with chronic abscessive pyoderma. No recurrences ofdisease activity were noted in patients with chronic recurrentosteofolliculitis, folliculitis, and furunculosis. . Aggravation of thedisease process was observed in one patient with an ulcerative form ofchronic pyoderma, i.e., manifested as an increase in the quantity ofpurulent discharge. No side effects or allergic reactions were observedin these studies.

Summary: The three step method of treatment, i.e., 1) treating withL-Glu-L-Trp, 2) assessing antibiotic sensitivity of isolates frompatients, and 3) selecting and administering an antibiotic to which thebacteria is sensitive, permitted the attending physician to choose foreach patient an individual antibiotic with highest anti-bacterialactivity. Seventy-eight percent of patients (78%) at the conclusion ofStage 1 showed a marked and reliable decrease in the microbialinhibitory concentration (MIC) of antibiotic required to inhibit growthof the isolated bacteria in vitro. In vivo, increased antibioticsensitivity was clinically manifested in 55-75% of the patients studied.At the conclusion of Stage 2 the combined treatment regimen withantibiotic and L-Glu-L-Trp resulted in complete recovery of 27previously incurable patients. In addition, 8 patients showedsignificant improvement and 1 showed moderate improvement.

While somewhat unexpected and provocative, the clinical outcome is notwithout a variety of possible scientific explanations. At least thefollowing possibilities come to mind: i) stimulation of cell mediatedimmunity in chronic staphylococcal infection leads to immuneelimination, or slower growth, of the more resistant populations ofbacteria; or, ii) L-Glu-L-Trp treatment induces production of a cytokinesuch as ASF (supra) or of lysozyme release at sites of infection; or,iii) immune mechanisms stimulated by L-Glu-L-Trp induce adown-regulation of the multiple drug resistant phenotype permitting ashift to more antibiotic sensitive bacteria in patients; or, iv)L-Glu-L-Trp may exert a direct antibacterial effect on Staphylococci.

Protocol A2: Chronic Staphylococcal Infection:

Summary Overview: L-Glu-L-Trp was administered to 52 patients sufferingfrom chronic skin diseases caused by antibiotic-resistant Staphylococci.42 patients with the same pathology but not treated with theimmunomodulator were the control group. L-Glu-L-Trp was administered imto 27 patients single daily at 100 μg for 5 days and intranasally to 25patients with the same daily and total dose. Differences between thesetwo methods of application were not noticed. The results of antibioticsensitivity testing showed that in all the patients with signs ofsecondary T-immunodeficiency, the antibiotic-sensitivity of thepatients' Staphylococci to one, a few, or all antibiotics was increasedsharply (more than 100-fold). The increased antibiotic sensitivitypermitted the physician to choose an antibiotic for each patient thathad previously shown a high level of activity for the bacteria. As awhole, within each group of patients there was a marked and reliableincrease in antibiotic sensitivity, i.e, decreased microbial inhibitoryconcentration (MIC) for all antibiotics studied. The treatment regimenwith L-Glu-L-Trp, followed by antibiotic sensitivity testing, and thenadministration of an individualized antibiotic provided completerecovery in all patients with previously incurable antibiotic resistantstaphylococcal infections.

Protocol A3: Chronic Staphylococcal Infection and Systemic LupusErythematosus: Patient Groups:

Group 1 constituted 50 patients with diagnosed.pyoderma: i.e., 29 malesand 21 females ranging from 17 to 59 years of age. All patients in thistrial had recurrent episodes of pyoderma and all had previously receivedconventional therapy including use of antibiotics, without effect.Length of illness was from 6 months to 16 years. Twenty-four of thepatients had one (or several) intermittent opportunistic and infectiousdiseases including: chronic tonsillitis (2), chronic maxillofacialsinusitis and sinusitis (2), dental granuloma (2), parodontitis (2),chronic cholestitis and pancreatitis (7), chronic gastritis andgastroenteritis (6), chronic bronchitis (1), chronic prostatitis (1),chronic lymphadenitis (2), chronic otitis (1), and focal pulmonarytuberculosis (1).

Group 1A (a subgroup of Group 1) consisted of 32 patients (17 males and15 females ranging from 17 to 42 years of age) with chronic pyodermalskin diseases including furunculosis, disseminated impetigo, multipleecthymas, recurring osteofolliculitis, seborrhea adiposa, and concreteseborrhea complicated by vulgar or abscessing acne. Diseases werecharacterized by a chronic course or frequent relapses, and at the timeof presentation for entry into the trial, all patients had receivedseveral prior courses of antibiotic therapy, none of which led to anydetectable clinical improvement. Patients in Group 1A were randomlydivided into two treatment groups, the first consisting of 16 patientstreated with L-Glu-L-Trp according to Therapy A, below, and the secondconsisting of 16 patients treated (as a control) according toconventional therapeutic methods- i.e., Therapy B (below). The resultsobtained with this patient population are summarized in the Tables,below.

Group 1B consisted of 18 patients (12 males and 6 females ranging in agefrom 27 to 62 years of age) having atypical forms of pyoderma persistingover 2-15 years and marked by chronic and recurrent courses with extremeresistance to therapy. Patients in Group 1B were randomly divided intotwo treatment groups, the first consisting of 10 patients (7 males and 3females) treated with L-Glu-L-Trp according to Therapy A, below, and thesecond consisting of 8 patients (5 males and 3 females) treated (as acontrol) according to conventional therapeutic methods; i.e., Therapy B(below). The results obtained with this patient population aresummarized in the Tables, below.

Group 2 constituted 22 patients with diagnosed forms of systemic lupuserythematosus (SLE): i.e., 14 males and 8 females ranging in age from 21to 62 years of age. The time since presentation with a diagnosis of SLEin this patient population ranged from several months to 18 years. AllSLE patients in this trial had dermal manifestations of lupuserythematosus that included discoid disseminated forms of the disease.All patients had experienced chronic and recurrent episodes of pyoderma,and all had previously received conventional therapy, including use ofantibiotics, delagil, and external corticosteroid ointments withouteffect. Patients in Group 2 were randomly divided into two treatmentgroups, the first consisting of 13 patients (9 men and 4 women) treatedwith L-Glu-L-Trp according to Therapy A, below, and the secondconsisting of 9 patients (5 men and 4 women) treated (as a control)according to Therapy B, ie., conventional therapeutic methods usinganti-malarial preparations and vasoactive agents according to standarddosages and schedules.

Treatment Protocols:

Therapy A: L-Glu-L-Trp was administered im to all patients at a dosageof 100 μg in a volume of 1 mL daily for 5 days. In treatment Protocol A,all patients received L-Glu-L-Trp in combination with antibiotics.

Therapy B (conventional therapy control): All patients in this protocolreceived the same antibiotics as in Therapy A and a nonspecificstimulatory agent, i.e., Thymogenin instead of L-Glu-L-Trp.

Laboratory Tests: Clinical and laboratory parameters of disease activitywere monitored including morning body temperature, leukocyte anddifferential blood cell count, hematology measurements and bloodchemistry and immunology determinations including the percentages andabsolute numbers of B- and T-lymphocytes, T-helper and T-suppressorcells, and in vitro blast responsiveness of the cells to staphylococcalprotein antigen, Concanavalin A mitogen, and staphylococcal protein A.Antibiotic sensitivity of Staphylococci was evaluated in patients beforeand after treatment with L-Glu-L-Trp or conventional antibioticsincluding penicillin (1), ampicillin (5), oxacillin (13), monomycin (1),cisomycin (1), tetracycline (9), erythromycin (3), garamycin (1),lincomycin (1), cephalosporin (2), Kefzol (1), Keflin (1), and cephalex(1). Local antibiotics were administered as follows: ampicillin (4),oxacillin (4), Levomycetin (1), tetracycline (9), Rondomycin (2),deoxycycline (8), cephalexin (2), and Rifampicin (1). The hematology andclinical chemistry laboratory test results obtained with the patients inGroup 1A, Group 1B, and Group 2 may be summarized as follows:

1. Hematology values were within the normal range of values prior totreatment and no significant differences were observed between the pre-and post-treatment values (i.e., either conventional therapy orL-Glu-L-Trp treatment) for clotting time of citrated whole blood thatwas recalcified; prothrombin clotting time; Thrombotest units; or,fibrinogen concentration in plasma; and,

2. Blood protein values were within the normal range prior to and aftertherapy for complement protein C3, alkaline phosphatase, bilirubin,creatinine, glucose, total protein, and concentrations of chloride,calcium, and sodium.

The results of laboratory tests of immune parameters of Groups 1A and 1Bare presented in TABLE 26.

                                      TABLE 26                                    __________________________________________________________________________    Differential Cell Counts and Immunology Values:                               Group 1A- Chronic Pyoderma; Group 1B- Atypical Pyoderma                                    Group 1A            Group 1B                                             Normal                                                                             Before                                                                              Control                                                                             L--Glu--L--Trp                                                                        Before                                                                             Control                                                                            L--Glu--L--Trp                             Healthy                                                                            Therapy                                                                             Therapy                                                                             Therapy Therapy                                                                            Therapy                                                                            Therapy                            Index   Values                                                                             (n = 32)                                                                            (n = 16)                                                                            (n = 16)                                                                              (n = 18)                                                                           (n = 8)                                                                            (n = 10)                           __________________________________________________________________________    Leukocytes                                                                    (× 10.sup.9 /L):                                                                6.7 ± 2                                                                          7.7 ± 1                                                                          7.9 ± 1                                                                           8.7 ± 0.5                                                                         6.5 ± 1                                                                         6.4 ± 1                                                                         6.2 ± 1                        % Normal Value:                                                                       (100%)                                                                             (115%)                                                                              (118%)                                                                              (130%)  (97%)                                                                              (96%)                                                                              (93%)                              Lymphocytes                                                                   %:      41.1 ± 12                                                                       27.3 ± 2                                                                         25.7 ± 2                                                                           25.9 ± 2.2                                                                       34.8 ± 3                                                                        34.7 ± 4                                                                        34.5 ± 4                        (× 10.sup.9 /L):                                                                2.8 ± 1                                                                           1.9 ± 0.1                                                                        2.2 ± 0.1                                                                      2.0 ± 0.2                                                                          2.26 ± 0                                                                        2.26 ± 0                                                                        2.14 ± 0                        % Normal Value:                                                                       (100%)                                                                             (68%) (79%) (71%)   (81%)                                                                              (81%)                                                                              (76%)                              T-Lymphocytes                                                                 (%):    61.4 ± 13                                                                       49.9 ± 7                                                                         55.9 ± 4                                                                         52.3 ± 5                                                                           63.5 ± 7                                                                        61.4 ± 7                                                                        55.1 ± 6                        (× 10.sup.9 /L):                                                                1.7 ± 1                                                                         1.13 ± 0                                                                         1.32 ± 0                                                                         1.15 ± 0                                                                           1.12 ± 0                                                                        1.21 ± 0                                                                        0.87 ± 0                        % Normal Value:                                                                       (100%)                                                                             (66%) (78%) (68%)   (66%)                                                                              (71%)                                                                              (51%)                              T-helper                                                                      (%):    47.1 ± 16                                                                       55.3 ± 6                                                                         48.3 ± 5                                                                           45.4 ± 4.9                                                                         39 ± 0                                                                        36.2 ± 6                                                                        32.1 ± 3                        (× 10.sup.9 /L):                                                                1.32 ± 1                                                                        1.07 ± 0                                                                         1.03 ± 0                                                                           0.92 ± 0.09                                                                      0.68 ± 0                                                                        0.65 ± 0                                                                        0.53 ± 0                        % Normal Value:                                                                       (100%)                                                                             (81%) (78%) (70%)   (52%)                                                                              (49%)                                                                              (40%)                              T-suppressor                                                                  (%):    14.3 ± 12                                                                       13.7 ± 3                                                                         21.3 ± 4                                                                         28.2 ± 4                                                                           24.5 ± 5                                                                        24.5 ± 5                                                                        23.1 ± 5                        (× 10.sup.9 /L):                                                                0.39 ± 0                                                                        0.31 ± 0                                                                         0.36 ± 0                                                                         0.51 ± 0                                                                           0.55 ± 0                                                                        0.54 ± 0                                                                        0.49 ± 0                        % Normal Value:                                                                       (100%)                                                                             (79%) (92%) (131%)  (141%)                                                                             (138%)                                                                             (126%)                             B-Lymphocytes                                                                 (%):      18 ± 11                                                                         7 ± 3                                                                            8 ± 2                                                                            7 ± 3                                                                              15 ± 3                                                                          11 ± 7                                                                         8.5 ± 1                        (× 10.sup.9 /L):                                                                0.49 ± 0                                                                        0.15 ± 0                                                                         0.14 ± 0                                                                         0.15 ± 0                                                                           0.25 ± 0                                                                        0.21 ± 0                                                                        0.14 ± 0                        % Normal Value:                                                                       (100%)                                                                             (31%) (29%) (31%)   (51%)                                                                              (43%)                                                                              (29%)                              IgG (g/L)                                                                              11 ± 2                                                                         15.1 ± 1                                                                         16.8 ± 1                                                                         16.2 ± 1                                                                             15 ± 4                                                                          15 ± 2                                                                        16.6 ± 2                        % Normal Value:                                                                       (100%)                                                                             (140%)                                                                              (156%)                                                                              (150%)  (141%)                                                                             (142%)                                                                             (154%)                             IgA (g/L)                                                                               2 ± 1                                                                           3 ± 0                                                                            3 ± 0                                                                          2.71 ± 1                                                                             2 ± 0                                                                           2 ± 0                                                                         2.21 ± 0                        % Normal Value:                                                                       (100%)                                                                             (156%)                                                                              (141%)                                                                              (143%)  (114%)                                                                             (124%)                                                                             (117%)                             IgM (g/L)                                                                               2 ± 0                                                                           1 ± 0                                                                            1 ± 0                                                                            1 ± 0                                                                              1 ± 1                                                                           1 ± 0                                                                         1.64 ± 0                        % Normal Value:                                                                       (100%)                                                                             (67%) (58%) (65%)   (84%)                                                                              (89%)                                                                              (104%)                             Erythrocyte                                                                   (sed. rate mm):                                                                       --   11.2 ± 1                                                                         12.9 ± 1                                                                         13.1 ± 1                                                                           --   --   --                                 Erythrocytes                                                                          --                                                                    (10.sup.12 /L):                                                                              4.3 ± 0.2                                                                        4.3 ± 0.2                                                                        4.2 ± 0.1                                                                         4.0 ± 0                                                                         4.0 ± 0                                                                          4.1 ± 0.1                     Neutrophils (%):                                                                      49.9 ± 12                                                                       62.4 ± 5                                                                         62.8 ± 5                                                                         63.8 ± 4                                                                           65.1 ± 5                                                                        67.8 ± 5                                                                          66.5 ± 3.2                    Monocytes (%)                                                                         --    5.9 ± 1                                                                          4.9 ± 2                                                                          4.5 ± 1                                                                            3.1 ± 2                                                                         2.9 ± 2                                                                          2.3 ± 1.6                     Basophils (%)                                                                         --    0.2 ± 0                                                                          0.2 ± 0                                                                         0.01 ± 0                                                                            0.1 ± 0                                                                         0.1 ± 0                                                                         0.1 ± 0                        Eosinophils (%)                                                                       --    3.1 ± 1                                                                          4.6 ± 1                                                                          4.2 ± 1                                                                            0.3 ± 2                                                                         0.4 ± 1                                                                         0.5 ± 1                        __________________________________________________________________________     †, statistically significant when compared with the pretreatment       values, i.e. p < 0.05.                                                   

                                      TABLE 27                                    __________________________________________________________________________    Lymphocyte Functional Activity:                                               Group 1A-Chronic Pyoderma; Group 1B- Atypical Pyoderma                                      Group 1A          Group 1B                                      Blast Transform.                                                                       Normal                                                                             Before                                                                             Control                                                                            L--Glu--L--Trp                                                                        Before                                                                             Control                                                                            L--Glu--L--Trp                      (Antigen)                                                                              Values                                                                             Therapy                                                                            Therapy                                                                            Therapy Therapy                                                                            Therapy                                                                            Therapy                             __________________________________________________________________________    PHA (% of PBL)                                                                         35.9 ± 21                                                                       49.1 ± 7                                                                        43.8 ± 7                                                                        42.7 ± 7                                                                           61.2 ± 23                                                                       72.3 ± 12                                                                       87.5 ± 5                         Con-A (% of PBL)                                                                       49.4 ± 21                                                                       72.9 ± 12                                                                       66.3 ± 12                                                                       62.8 ± 8                                                                           52.5 ± 25                                                                       69.4 ± 12                                                                       96.1 ± 4                         Staph-A (% of PBL)                                                                     95.3 ± 12                                                                       75.3 ± 10                                                                       79.4 ± 10                                                                       85.6 ± 9                                                                           87.5 ± 4                                                                        82.5 ± 5                                                                        75.3 ± 1                         __________________________________________________________________________     (No mean values were significantly different than the Pretreatment values     i.e. p > 0.05)                                                           

Summary of Clinical Disease Responses in the Patients in Group1A-Chronic Pyoderma: The results presented in TABLE 27 show thattreatments with L-Glu-L-Trp increased the percentage of lymphocytesblast-transformed following in vitro incubation in media containing PHAor Con-A when isolated from patients with diagnosed atypical pyoderma,but not from patients with chronic pyoderma.

Summary of Clinical Disease Responses in the Patients in Group1B-Atypical Pyodenna: The clinical investigators conducting this trialconcluded as follows: "A clinical analysis of the conducted treatmentshow that, in comparison with the traditional therapy methods, noimportant advantages in using L-Glu-L-Trp were found. Thus, L-Glu-L-Trptreatment did not cause normalization of the immunologic changes foundin this group of patients with chronic atypical pyoderma and did notturn out to be effective clinically."

The results obtained in the patients of Group 2 are summarized below inTABLES 28-29.

                                      TABLE 28                                    __________________________________________________________________________    Differential Cell Counts and Immunology Values:                               Group 2: Pyoderma in Patients with SLE                                                        Before Conventional                                                                         L--Glu--L--Trp                                                  Therapy                                                                              Therapy                                                                              Therapy                                         Index    Normal Range                                                                         (n = 22)                                                                             (n = 9)                                                                              (n = 13)                                        __________________________________________________________________________    Leukocytes                                                                    (× 10.sup.9 /L):                                                                 6.7 ± 2.4                                                                         6.15 ± 0.23                                                                       6.75 ± 0.3                                                                        5.74 ± 0.12                                  % Normal Value:                                                                        (100%) (92%)  (101%) (86%)                                           Lymphocytes                                                                   %:       41.4 ± 11.9                                                                       33.1 ± 1.6                                                                        33.9 ± 1.7                                                                        36.4 ± 2.3                                   (× 10.sup.9 /L):                                                                 2.8 ± 1.4                                                                         2.11 ± 0.12                                                                       2.13 ± 0.21                                                                       2.17 ± 0.21                                  % Normal Value:                                                                        (100%) (75%)  (76%)  (78%)                                           T-Lymphocytes                                                                 (%):     61.4 ± 13.1                                                                       48.9 ± 4.3                                                                        42.5 ± 3.1                                                                        44.3 ± 2.7                                   (× 10.sup.9 /L):                                                                 1.7 ± 0.9                                                                         1.02 ± 0.08                                                                       0.63 ± 0.08                                                                       1.13 ± 0.07                                  % Normal Value:                                                                        (100%) (60%)  (37%)  (66%)                                           T-helper                                                                      (%):     47.1 ± 16.2                                                                       53.2 ± 1.9                                                                        44.3 ± 3.4                                                                        26.5 ± 2.7                                   (× 10.sup.9 /L):                                                                 1.32 ± 0.81                                                                       1.08 ± 0.15                                                                       1.26 ± 0.15                                                                       .sup. 0.87 ± 0.12.sup.†               % Normal Value:                                                                        (100%) (82%)  (95%)  (66%)                                           T-suppressor                                                                  (%):     14.3 ± 12.3                                                                       12.4 ± 2.1                                                                        9.3 ± 5.1                                                                         21.2 ± 3.7.sup.††              (× 10.sup.9 /L):                                                                 0.39 ± 0.38                                                                       0.29 ± 0.06                                                                       0.26 ± 0.07                                                                       .sup. 0.53 ± 0.03                            % Normal Value:                                                                        (100%) (74%)  (67%)  (136%)                                          B-Lymphocytes                                                                 (%):     18.1 ± 11                                                                         13.5 ± 1.01                                                                       12.1 ± 0.8                                                                        6.8 ± 0.9                                    (× 10.sup.9 /L):                                                                 0.49 ± 0.3                                                                        0.31 ± 0.02                                                                       0.26 ± 0.01                                                                       0.16 ± 0.01                                  % Normal Value:                                                                        (100%) (63%)  (53%)  (52%)                                           IgG (g/L)                                                                              10.8 ± 2.13                                                                       13.9 ± 1.34                                                                       10.2 ± 1.39                                                                       14.8 ± 1.27                                  % Normal Value:                                                                        (100%) (128%) (94%)  (137%)                                          IgA (g/L)                                                                              1.90 ± 0.99                                                                       2.21 ± 0.41                                                                       1.54 ± 0.52                                                                       2.85 ± 0.32                                  % Normal Value:                                                                        (100%) (116%) (81%)  (150%)                                          IgM (g/L)                                                                              1.58 ± 0.47                                                                       1.28 ± 0.47                                                                       0.72 ± 0.26                                                                       0.92 ± 0.12                                  % Normal Value:                                                                        (100%) (81%)  (46%)  (58%)                                           Erythrocytes                                                                  (10.sup.12 /L): 4.4 ± 0.1                                                                         4.3 ± 0.1                                                                         4.7 ± 0.1                                    Neutrophils                                                                   (%):     49.9 ± 11.9                                                                       56.8 ± 2.8                                                                        59.7 ± 3.7                                                                        55.3 ± 4.2                                   (× 10.sup.9 /L):                                                                 3.3 ± 1.1                                                                         2.6 ± 1.1                                                                         1.8 ± 1.2                                                                         3.5 ± 0.7                                    % Normal Value:                                                                        (100%) (79%)  (55%)  (106%)                                          Monocytes (%)   6.0 ± 0.9                                                                         5.0 ± 0.8                                                                         8.0 ± 0.7                                    Basophils (%)   0.1 ± 0.1                                                                         0.1 ± 0.1                                                                         0.1 ± 0.1                                    Eosinophils (%) 2.7 ± 0.2                                                                         1.8 ± 0.2                                                                         2.2 ± 0.1                                    __________________________________________________________________________     .sup.†† statistically significant in comparison with the        pretreatment values, i.e., p < 0.01                                           .sup.† statistically significant in comparison with the                pretreatment values, i.e., p < 0.05                                      

                                      TABLE 29                                    __________________________________________________________________________    Lymphocyte Functional Activity:                                               Group 2- Pyoderma in Patients with SLE                                                   Normal                                                                              Before                                                                              Conventional                                                                         L--Glu--L--Trp                                  Blast Transformation                                                                     Healthy                                                                             Therapy                                                                             Therapy                                                                              Therapy                                         (Antigen)  Values                                                                              (n = 22)                                                                            Control (n = 9)                                                                      (n = 13)                                        __________________________________________________________________________    PHA (% of PBL)                                                                           35.9 ± 20.7                                                                      58.1 ± 4.0                                                                       47.1 ± 3.7                                                                        47.4 ± 3.5                                   Con-A (% of PBL)                                                                         49.4 ± 20.7                                                                      57.2 ± 7.9                                                                       58.1 ± 8.3                                                                        64.1 ± 6.5                                   __________________________________________________________________________     (No mean values were significantly different than the Pretreatment values     i.e. p > 0.05)                                                           

Summary of Clinical Disease Responses in the Patients in Group 2--SLEPatients with Pyoderma: The investigators conducting the trialconcluded: L--Glu--L--Trp "treatment proved more rational and adequatein the plan of correcting the immune shift discovered. Treating withL--Glu--L--Trp leads to a positive clinical effect and resolution ofdermal rashes. Treatment with delagil in combination with vascularagents" (i.e., traditional therapy) "leads to the appearance ofadditional deviations in the immunity system and requires mandatoryexternal treatment with corticosteroids."

Protocol B: Psoriasis

Summary Overview: A total of 30 patients with psoriasis were treatedwith L--Glu--L--Trp (Group 1) and 30 other psoriasis patients wereentered into a control group (Group 2) and treated using conventionaltreatment. All patients had at least a 5 year history of unsuccessfulantibiotic therapy. The administration of 100 μg im or 1 μg/kgintranasally daily for a period of 10 days resulted in the improvementin 7% of the patients, significant improvement in 60% of patients, andtotal recovery in 33% of the patients. Patients with psoriasis in theprogressive phase exhibited i) a depression of cell mediated immuneparameters, ii) an increase in serum concentrations of IgG, IgA, andIgM, iii) hyper-coagulation, and, iv) depressed fibrinolytic activity.

Patient Groups: Group 1 constituted 30 patients, aged 15-60 years andwith length of illness in most patients being>5 years. In 28 of the 30patients, the illness was of a progressive nature, and in 29 of the 30,relapses had been observed during the winter months. Group 2 constituted30 patients with disseminated forms of psoriasis.

Trial Protocols: Treatment B1: Traditional therapy in combination withL--Glu--L--Trp was administered to all patients in Group 1: i.e.,L--Glu--L--Trp was administered im at a dosage of 100 μg in a volume of1 mL with 0.25% novocaine every other day for 10 days. Treatment B2:Traditional therapy only was administered to the patients in Group 2:i.e., sedative desensitizers, vitamins A, B₆, and B₁₂, and folic acid,pyrogenic preparations, sedatives, UFO, and local prescription ofsalidol ointment and 3-5% sulfur salicylic and/or corticosteroid(salidol) ointments. Some modifications of treatment regimens wererequired because of intolerance to B group vitamins in three cases whichmanifested itself in dermal puritus and urticarial eruptions.

Laboratory parameters of disease activity were monitored includingmorning body temperature, leukocyte and differential blood cell count,hematology measurements and blood chemistry, acute phase proteins, andimmunology determinations including the percentages and absolute numbersof B- and T-lymphocytes. Comparisons were made to laboratory valuesobtained from normal healthy persons aged 18 to 40 years.

Clinical parameters of disease activity were monitored by grading dermalmanifestations of disease as follows: "improvement" defined asstabilization of the dermal disease process and lightening and smoothingof papular, plaque, and rash eruptions; "considerable improvement"defined as indications of dermal locations having maculas, and/orsolitary smooth papules having a pale color, or resembling "maternalplaques," i.e., birth marks; and, "recovery" defined as indications ofdermal locations having eruptions or maculas that are depigmented ormildly pink in color.

The findings of laboratory investigations are summarized in TABLES30-32.

                                      TABLE 30                                    __________________________________________________________________________    Hematology Values                                                                           Healthy                                                                             Patients                                                                             Conventional                                                                         L--Glu--L--Trp                                            Normal                                                                              Before Therapy                                                                              Therapy                                                   Controls                                                                            Therapy                                                                              Group 2                                                                              Group 1                                     Index         (n = 20)                                                                            (n = 40)                                                                             (n = 30)                                                                             (n = 30)                                    __________________________________________________________________________    Clotting time (citrated whole                                                               123 ± 2.1                                                                        102 ± 3.1**                                                                       135 ± 3.3.sup.††                                                    141 ± 2.4.sup.††           blood-recalcified)                                                            Kaolin-Cephalin clotting time                                                               53.6 ± 0.72                                                                      49.8 ± 0.8**                                                                       57 ± 0.4.sup.††                                                     58 ± 0.3.sup.†                   PT             22 ± 0.3                                                                        21 ± 0.37                                                                         21.6 ± 0.4 .sup.                                                                   23 ± 0.2.sup.                           Thrombotest (units)                                                                         30.7 ± 0.56                                                                      29.8 ± 0.37                                                                        31 ± 0.7.sup.                                                                     33 ± 0.5.sup.††           Fibrinogen Concentration (g/L)                                                              2.74 ± 0.56                                                                      3.2 ± 0.37                                                                        .sup.  3.0 ± 0.36.sup.                                                             3.4 ± 0.1.sup.                          FDP (μg/mL)                                                                              --.sup.a                                                                            64 ± 2.2                                                                            45 ± 2.14.sup.††                                                   23 ± 1.8.sup.††           Fibrinolytic Activity (min.)                                                                 154 ± 12.6                                                                      200 ± 7.0*                                                                        196 ± 6.6.sup.                                                                    200 ± 2.7.sup.††           __________________________________________________________________________     *Statistically different than the normal control values, i.e., p < 0.05;      *, statistically different than the normal control values at the p < 0.00     level;                                                                        .sup.†, statistically different than the pretreatment values, i.e.     p < 0.05,                                                                     .sup.††, statistically different than the pretreatment          values, p < 0.001;                                                            a. FDP, fibrin degradation products are usually not detectable in normal      plasma if clotting is properly inhibited.                                

Summary of Treatment Effects on Hemostatic Values

Conventional Therapy: Shifts in certain coagulation parameters afterconventional therapy, although significant, were not pronounced. Slightchanges were observed in patients treated with conventional therapy asfollows: i) in positive reactions of clotting to ethanol (data not shownabove), ii) a slower clotting time for recalcified plasma, and (iii) aslightly slower clotting time for kaolin-cephalin induced clotting(KCT). Fibrinopeptide (FDP) concentration, (an indicator of the level ofongoing fibrinolysis in patients), decreased insignificantly.

Patients receiving L--Glu--L--Trp exhibited the following changes incoagulation measurements: i) clotting time for recalcified plasma wasslowed, ii) KCT was also decreased, iii) thrombin time (Thrombotest;PTT) was decreased, iv) FDP concentration dropped significantly, v)antithrombin III (ATIII) decreased to normal levels, and vi)Hagemann-factor-dependent fibrinolytic activity was returned to normallevels. Overall, the changes appeared to indicate a decrease inunderlying thrombotic processes in the patients treated withL--Glu--L--Trp, possibly by activation of cellular fibrinolyticactivities, e.g., release of mediators inducingHagemann-factor-dependent fibrinolysis. (Similar effects on FDP andATIII were recorded in studies with patients in Example 6, Protocol C3,below; and, in the malaria patients of Example 19, also below.)

                                      TABLE 31                                    __________________________________________________________________________    Blood Acute-Phase Protein Measurements                                                     Normal        Conventional                                                                         L--Glu--L--Trp                                           Healthy                                                                             Before  Therapy                                                                              Therapy                                                  Values                                                                              Therapy Group 2                                                                              Group 1                                     Index        (n = 20)                                                                            (n = 40)                                                                              (n = 30)                                                                             (n = 30)                                    __________________________________________________________________________    C3 component (mcg/mL)                                                                      814 ± 46                                                                          893 ± 14.6                                                                        945 ± 14.8                                                                        926 ± 28.4                               Prealbumin (mcg/mL)                                                                          304 ± 11.5                                                                       224 ± 4.49**                                                                     248 ± 3.62                                                                        357 ± 3.8.sup.††           Ceruloplasmin (mcg/mL)                                                                     255 ± 16                                                                           329 ± 3.3**                                                                      350 ± 11.sup.††                                                     .sup. 366 ± 11.4.sup.††                                      4                                           Orosomucoid (mcg/mL)                                                                       706 ± 31                                                                           837 ± 12.8**                                                                     .sup. 977 ± 26.7.sup.††                                              829 ± 13.2.sup.                         α2-macroglobulin (mcg/mL)                                                             2.3 ± 0.82                                                                      2.3 ± 0.3                                                                          .sup. 2.5 ± 0.07                                                                  .sup. 2.5 ± 0.01                         Transferrin (mcg/mL)                                                                       3.11 ± 0.1                                                                       2.87 ± 0.06                                                                        2.74 ± 0.08                                                                       .sup. 3.36 ± 0.08.sup.†.dagger                                      .                                           __________________________________________________________________________     *Statistically different than the normal control values, i.e., p < 0.05;      **, statistically different than the normal control values at the p <         0.001 level;                                                                  .sup.††, statistically different than the pretreatment          values, i.e., p < 0.001.                                                 

Acute Phase Reactants: Acute phase reactants ceruloplasmin andorosomucoid were significantly elevated, and prealbumin was depressed,prior to treatment. Patients in Group 2 experienced a further increasein orosomucoid concentration during therapy, and other acute phasereactants, while not increasing significantly, did not dropsignificantly in this control group. In patients treated withL--Glu--L--Trp (Group 1) ceruloplasmin, transferrin, and prealbuminincreased significantly.

                                      TABLE 32                                    __________________________________________________________________________    Differential Cell Counts and Immunology Values                                                        Conventional                                                                        Combination                                              Normal Healthy                                                                        Before Therapy                                                                             L--Glu--L--Trp                                           Values  Therapy                                                                              Control                                                                             Therapy                                         Index    (n = 20)                                                                              (n = 40)                                                                             (n = 30)                                                                            (n = 30)                                        __________________________________________________________________________    Leukocytes                                                                    (× 10.sup.9 /L):                                                                  5.8 ± 0.25                                                                         6.6 ± 0.1**                                                                      6.7 ± 0.1                                                                         9.0 ± 1.4.sup.††              % Normal Value:                                                                        (100%)  (114%) (116%)                                                                              (155%)                                          Lymphocytes                                                                   (× 10.sup.9 /L):                                                                 1.74 ± 0.12                                                                        1.9 ± 0.07                                                                         1.7 ± 0.03                                                                       2.5 ± 0.09.sup.††             % Normal Value:                                                                        (100%)  (109%) (98%) (144%)                                          T-Lymphocytes                                                                 (× 10.sup.9 /L):                                                                 0.89 ± 0.06                                                                        0.55 ± 0.04*                                                                      0.50 ± 0.01                                                                      0.94 ± 0.07.sup.††             % Normal Value:                                                                        (100%)  *      (56%) (106%)                                                           (62%)                                                        B-Lymphocytes:                                                                (× 10.sup.9 /L):                                                                 0.46 ± 0.02                                                                        0.36 ± 0.01*                                                                      0.32 ± 0.02                                                                      0.53 ± 0.02.sup.††             % Normal Value:                                                                        (100%)  *      (69%) (115%)                                                           (78%)                                                        IgG (mg/mL)                                                                             134 ± 5.6                                                                          143 ± 7.3                                                                         146 ± 4.4                                                                        187 ± 1.9 .sup.†                     % Normal Value:                                                                        (100%)  (107%) (109%)                                                                              (140%)                                          IgA (mg/mL)                                                                            98.3 ± 6.2                                                                          140 ± 5.1**                                                                       159 ± 5.1                                                                        138 ± 6.8 .sup.                             % Normal Value:                                                                        (100%)  (142%) (162%)                                                                              (140%)                                          IgM (mg/mL)                                                                             127 ± 9.7                                                                         153 ± 5.9*.sup.                                                                    179 ± 4.0                                                                        193 ± 9.6 .sup.                             % Normal Value:                                                                        (100%)  (120%) (141%)                                                                              (152%)                                          __________________________________________________________________________     *Statistically different than the normal control values, i.e., p < 0.05;      **, statistically different than the normal control values at the p <         0.001 level;                                                                  .sup.†, statistically different than the pretreatment values, i.e.     p < 0.001.                                                               

Cellular Immune Parameters: In the trial group of patients withpsoriasis there was a slight increase in lymphocyte count and asignificant (p<0.001) decrease in T- and B-lymphocytes. Serumimmunoglobulins were also elevated, i.e., IgA, IgM, and to a lesserextent IgG. After L--Glu--L--Trp treatment, a considerable increase wasobserved in the number of leukocytes and lymphocytes. The quantity of T-and B-lymphocytes approached normal and the number of T-helper cellsalso increased. The number of T-suppressors increased less dramatically.The levels of IgG and IgM after therapy remained unchanged, and IgAdecreased, yet did not reach normal. Thus, employing L--Glu--L--Trp incombined therapy led to significant increases in peripheral bloodlymphocytes and T-lymphocytes in this psoriasis patient group.

Clinical Responses: Both conventional and L--Glu--L--Trp treatmentsresulted in remission of disease activity by day 8-10 (TABLE 33) inapproximately the same percentage of patients. However, patients inGroup 1 showed improvement more rapidly, i.e., with only the secondinjection of L--Glu--L--Trp there was noted an apparent pallor ofpsoriatic eruptions as well as a gradual smoothing of eruptions.Overall, disease activity in the combination L--Glu--L--Trp-treatedpatients resolved 2-3 days earlier than in patients treated with onlythe conventional therapy, and this was most evident as i) a more rapiddecrease in erythema of the skin eruptions (i.e., 2.8 days sooner thancontrols), ii) a more rapid smoothing of papules and plaques, and iii) amore rapid disappearance of psoriatic rash (i.e., 7.1 days sooner thancontrols). The length of hospital stay for patients in Group 1 was alsoan average of 4.2 days shorter than patients in Group 2. Clinicalevaluation of patients' disease activity at day 8-10 is summarized inthe following TABLE 33.

                  TABLE 33                                                        ______________________________________                                        Clinical Evaluation of Patients Response to Therapy                                   Number of Patients Having a Clinical Outcome as:                      Treatment No                  Considerable                                    Group     Improvement                                                                             Improvement                                                                             Improvement                                                                           Recovery                                ______________________________________                                        Conventional                                                                            1/30      6/30      16/30   7/30                                    Treatment (3%)      (20%)     (55%)   (23%)                                   (Group 2)                                                                     L--Glu--L--Trp                                                                          3/30      2/30      18/30   7/30                                    Therapy   (10%)     (7%)      (60%)   (23%)                                   (Group 1)                                                                     ______________________________________                                    

Dermatological Diseases (continued)

Protocol C: Wound Healing

Summary Overview: L--Glu--L--Trp was administered to 37 patients withwounds of various origins, types and localizations. Patients in thecontrol group (24) received conventional treatments. L--Glu--L--Trp wasadministered daily im (or topically) on each of 10 consecutive days. Theim dose was 100 μg. L--Glu--L--Trp accelerated wound healing (incomparison to the controls.), and L--Glu--L--Trp treatment reduced theduration of therapy and prevented development of infectiouscomplications.

Protocol D: Burns

Summary Overview: A total of 23 patients with cutaneous burns weretreated with L--Glu--L--Trp either im or intranasally. Fourteen patientsin a control group were treated using conventional methods. Individualstreated with L--Glu--L--Trp exhibited an accelerated rate of woundhealing, a diminished frequency of infections, and less scarring wasobserved.

Protocol E: Frostbite

Summary Overview: Seventeen patients with frostbite of the extremitieswere treated with L--Glu--L--Trp either im or intranasally. Elevenpatients constituted a control group. Rapid healing and restoration oftissue integrity was observed in patients receiving L--Glu--L--Trptreatment.

EXAMPLE 7 Obstetric and Gynecologic Diseases

Protocol A: Pelvic Inflammatory Diseases

Summary Overview: Ninety-six female patients (96) were entered into atrial having a variety of different disorders, i.e., pelvic inflammatorydiseases, cervicitis, vaginitis and various tubo-ovarian and adnexalabscesses. Forty-six patients (46) comprised the L--Glu--L--Trptreatment group, and 50 patients comprised a conventional treatmentcontrol group. L--Glu--L--Trp was administered daily im at a dose of 100μg, or 1 μg/kg intranasally, on each of 5 consecutive days.Alternatively, 50 μg of L--Glu--L--Trp was injected intralymphaticallyon each of 5 consecutive days in conjunction with conventional therapy.L--Glu--L--Trp treatment alleviated pain, reduced fever, and decreasedthe duration of the medical treatment in comparison with the controlgroup receiving conventional treatment. Normalization of immuneparameters was correlated with the observed clinical improvements.

Patient Populations: The patient population constituted 96 acutely illpatients (18 to 50 years of age): 46 were treated with L--Glu--L--Trp(Group 1), and 50 were treated using conventional antibiotic therapy(Group 2). Patients entered into Group 1 (aged 19-50) were separableinto clinical subgroups as follows: Subgroup 1A, consisted of 18patients exhibiting febrile endometriosis (body temperature 38°-39° C.),and suppurative vaginal discharge after infected abortions performedoutside hospitals (average age 24.4±4.2 years); Subgroup 1B, consistedof 16 patients with abdominal pains resulting from chronic nonspecificsubfebrile (<38° C.) inflammatory disease of the fallopian tubes(average age 32.2±3.9 years; range of abortions in the patientpopulation 2-8); and, Subgroup 1C consisted of 12 patients withinflammation of the fallopian tubes, (38% resulting from placement of anIUD in the uterus more than 4 years previously). All patients hadpreviously proved unresponsive to a full course of broad spectrumantibiotic therapy (i.e., metronidazole, nitrofurans, andsulfanilamides) delivered on an in-patient basis (average age 34.5±5.3years). Group 2 (control) was constituted of 50 patients (aged 18-44)presenting with analogous symptoms and diseases and the patients in thisgroup were treated with traditional antibiotic therapy.

Treatment Protocols: In all cases L--Glu--L--Trp was delivered im at adose of 100 μg in 1 ml volume daily on each of five days. Treatment A:Administered to patients in Group 1A: The course of L--Glu--L--Trptherapy was administered, remnants of fetal tissue were surgicallyremoved, and an additional course of L--Glu--L--Trp therapy wasadministered.

Treatment B: Administered to patients in Group 1B: L--Glu--L--Trptherapy was administered immediately upon admission into the protocol.

Treatment C: Administered to patients in Group 1C: Infected uterinetissues were surgically removed, abdominal drainage was established, andL--Glu--L--Trp therapy was administered starting 2 dayspost-operatively.

Laboratory Tests and Clinical Monitoring: Patients were evaluated bymonitoring the following parameters before, during, and 5-7 days aftertherapy: i) clinical status of the patient (i.e., body temperature,abdominal pain, and physical examinations) and, ii) laboratory data(i.e., urinalysis, blood chemistry, and blood immunology). The followingclinical chemistry values of the patients in Groups 1A, 1B, and 1C wereall within the normal range before and after treatment: AST, ALT,creatinine, urea, glucose, protein, chloride, potassium, and sodium.Patients in Groups 1A-1C and Group 2 exhibited elevated levels ofalkaline phosphatase (80±13 ED/ml) and bilirubin (1.0±0.05 mg %) priorto treatment, but the values were still within the normal range, i.e.,normal alkaline phosphatase (30-85 ED/ml), and normal bilirubin(0.15-1.0 mg %). Treatment with L--Glu--L--Trp reduced the latter serummarkers, i.e., alkaline phosphatase fell to 62.71±5.67 ED/mL andbilirubin to 0.50±0.03 mg %.

Laboratory immunology test results were not statistically differentbetween Groups 1A, 1B, and 1C before or after receiving Treatment A, B,and C, respectively, so the data were pooled and presented together inTABLE, 34.

                                      TABLE 34                                    __________________________________________________________________________    Immunology Values                                                                                      After                                                           Normal        Conventional                                                                         After                                                    Value  Before Antibiotic                                                                           L-Glu-L-Trp                                   Index      (Healthy)                                                                            Therapy                                                                              Therapy                                                                              Therapy                                       __________________________________________________________________________    Lymphocytes:                                                                  % of PBL:  41.11 ± 1.54                                                                      14.70 ± 1.26†                                                              19.09 ± 1.21*                                                                     36.61 ± 1.32*                              No. (10.sup.9 /L):                                                                       2.78 ± 0.14                                                                       1.36 ± 0.11†                                                               1.23 ± 0.09                                                                       2.26 ± 0.12*                               % Normal Value:                                                                          (100%) (49%)  (44%)  (81%)                                         T-Lymphocytes:                                                                % of Lym:  61.42 ± 1.98                                                                      76.44 ± 1.52                                                                      74.1 ± 1.68                                                                       71.57 ± 1.76                               No. (10.sup.9 /L):                                                                       1.69 ± 0.12                                                                       1.02 ± 0.11†                                                               0.75 ± 0.08*                                                                      1.67 ± 0.09*                               % of Normal Value:                                                                       (100%) (60%)  (38%)  (75%)                                         B-Lymphocytes:                                                                % of PBL:  18.14 ± 1.12                                                                      11.30 ± 1.06                                                                      8.91 ± 0.78                                                                       11.09 ± 0.87                               No. (10.sup.9 /L):                                                                       0.49 ± 0.08                                                                       0.17 ± 0.02†                                                               0.14 ± 0.02                                                                       0.27 ± 0.03*                               % of Normal Value:                                                                       (100%) (35%)  (28%)  (55%)                                         Phagocytes:                                                                   % PBL:     64.43 ± 1.94                                                                      76.30 ± 2.39                                                                      73.36 ± 2.38                                                                      74.78 ± 2.35                               IgM (g/L)  1.50 ± 0.17                                                                       1.90 ± 0.20                                                                       1.39 ± 0.10                                                                       2.64 ± 0.36*                               % Normal Value:                                                                          (100%) (126%) (93%)  (176%)                                        IgG (g/L)  10.84 ± 0.59                                                                      12.32 ± 0.75                                                                      12.91 ± 0                                                                         13.73 ± 0.51                               % Normal Value:                                                                          (100%) (114%) (119%) (126%)                                        IgA (g/L)  1.91 ± 0.15                                                                       1.89 ± 0.13                                                                       1.86 ± 0.1                                                                        2.61 ± 0.33*                               % Normal Value:                                                                          (100%) (99%)  (97%)  (137%)                                        Blastogenic Response:                                                         % Lym blast w/ PHA:                                                                      35.83 ± 2.82                                                                      64.13 ± 4.90†                                                              54.84 ± 4.4                                                                       37.22 ± 2.81*                              % Lym blast w/ Con A                                                                     49.4 ± 3.81                                                                       72.65 ± 5.61                                                                      62.89 ± 5.1                                                                       68.70 ± 5.32                               __________________________________________________________________________     †, a p value of <0.05 was recorded in these studies after              mathematical comparisons of Normal values with the Pretreatment values;       *, a p value of <0.05 was recorded in these studies after mathematical        comparisons of Normal values with the Pretreatment values.               

The results presented in TABLE 34, above, show that prior to therapy thepatients in this trial population had decreased absolute levels oflymphocytes (49% of normal), decreased T-lymphocytes (60% of normal),and decreased B-lymphocytes (35% of normal). On conventional therapy,the levels of all three monitors of immune function continued todeteriorate. In patients treated with L--Glu--L--Trp, the levels oflymphocytes increased to about 81% of normal, with T- and B-lymphocytesincreasing to about 75% and 55%, respectively.

Protocol B: Complications of Pregnancy

Summary Overview: One hundred fifty one patients (151) exhibitingtoxemia in the first or second half of pregnancy were treated witheither L--Glu--L--Trp (97 patients) or with conventional therapy (54patients). L--Glu--L--Trp was administered im at 100 μg daily, or 1μg/kg intranasally, for 5-10 days. L--Glu--L--Trp treatment normalizedblood pressure, peripheral edema was reduced, abnormal blood chemistryvalues prior to treatment returned to within the normal range, andimmune indices that were altered prior to treatment were returned towithin the normal range.

Protocol C Complications of Pregnancy

Summary Overview: L--Glu--L--Trp was administered to 34 pregnant women.27 pregnant women received conventional treatment served as a controlgroup. L--Glu--L--Trp was administered daily at a dosage of 100 μg im,or 1 μg/kg intranasally, on each of 5-10 days. Signs of clinicalimprovement were resolution of weakness and dizziness, increasedappetite, and the normalization of the immunological and hematologicalindices. Decreased fetal hypoxia was also observed in women treated withL--Glu--L--Trp.

Protocol D: Post-Partem Infections

Summary Overview: Nineteen patients (19 women) post-term were treatedwith L--Glu--L--Trp and 48 women received conventional post-termtreatment as a control group. Administration of 100 μg L--Glu--L--Trp im(or 1 μg/kg intranasally), over a period of 3-5 days resulted in theeffacement of the cervix with thinning of the cervix and the descent ofthe fetus, resulting in a normal delivery.

Protocol E: Uterine Infections

Summary Overview: L--Glu--L--Trp was administered in combination withantibiotic therapy to twelve female patients (average age 34) withchronic uterine infections caused by placement of I.U.D.'s (i.e., meanlength of infection 4-5 years; maximum 15 years). All patients hadreceived more than one previous course of antibiotic therapy during thecourse of their illness, and most patients had received several coursesof therapy. Conventional treatment consisted of surgical removal of theI.U.D. followed by a course of antibiotic therapy. The control groupconsisted of 50 patients with uterine infections, (same cause), treatedusing conventional therapy. L--Glu--L--Trp was administered withconventional therapy by injecting 100 μg im daily on each of fiveconsecutive days. Differences between the symptoms of post-operativerecovery of the patients in the L--Glu--L--Trp treatment group (i.e., incomparison with the control group) were noted within as little as 3-4days as increased appetite, and normalization of sleep and bodytemperature. Eleven of the 12 patients in the L--Glu--L--Trp treatmentgroup healed more rapidly than the patients in the control group andphysical therapy was started sooner than with the patients in thecontrol group. Intramuscular injections of L--Glu--L--Trp did not induceany visible side effect or allergic reaction.

EXAMPLE 8 Herpes Virus Infections

Protocol A: Herpes vulgaris

Summary Overview: Patients treated with L--Glu--L--Trp either topically,im, or intranasally experienced marked reduction of recurrence ofherpetic lesions, with substantial reduction in the period betweenoutbreaks. In one trial, individuals who experienced 7-10 outbreaks peryear experienced less than one outbreak per year after treatment withL--Glu--L--Trp in combination with interferon.

Protocol B: Herpes Zoster

Summary Overview: A total of 37 patients with Herpes Zoster were treatedwith L--Glu--L--Trp in combination with conventional interferontreatment. Twenty-five control patients (25) were treated withinterferon alone. Administration of L--Glu--L--Trp was as a single dailyinjection of 100 μg im, or 1 μg/kg intranasally, over a period of 10days. Treatment with L--Glu--L--Trp resulted in accelerated clearing offoci of herpes infection. Recurrence of lesions was prevented andhealing occurred on the average 40% earlier in the L--Glu--L--Trptreated group than in the control group. Changes in immunologicalindices in the L--Glu--L--Trp treated patients were correlated with thefavorable clinical outcome.

EXAMPLE 9 Dental Diseases

Protocol A: Gingivitis

Summary Overview: Patients were treated for gingival disease bysubcutaneous administration of L--Glu--L--Trp into the area of thegingiva. The treatment resulted in an arrest of gingival disease.Approximately 160 patients were studied, 80 patients were treated withL--Glu--L--Trp and an equal number were treated using conventionaltherapy (control). Administration of 100 μg L--Glu--L--Trp im,subcutaneously, or by electrophoresis (whereby a small voltage charge tothe gums results in a rapid transfer of medication through the gumepithelium) resulted in a more rapid arrest of bleeding, eliminated ofinflammation, and decrease in purulent discharge. L--Glu--L--Trptreatment resulted in fewer recurrences of gingival disease.Normalization of immunologic indices and coagulation in theL--Glu--L--Trp treated patients was correlated with favorable clinicaloutcome.

Protocol B: Dental Caries, Odontogenic Infections, Periapical GranulomasSummary Overview: Treatment of dental caries with toothpaste containingL--Glu--L--Trp may result in a reduction of caries. The use of dentaltoothpaste containing L--Glu--L--Trp may have the secondary effect ofreducing incidence and severity of gingival disease. Patients withodontogenic infections and periapical granulomas can be treated with 100μg of L--Glu--L--Trp injected into the foramen at the base of the tooth,or alternatively, L--Glu--L--Trp can be compounded in a filling pastewhich is packed into the base of the tooth.

Protocol C: Odontogenic Infections--Periapical Granulomas

Summary Overview: Forty-six patients (46; aged 3-14 years) withperiapical granulomas and limited, focal, and diffuse forms of purulentodontogenic osteomyelitis were treated with L--Glu--L--Trp. Twenty-eightpatients (15) having the same disease profile and under conventionaltreatment formed a control group.

Patient Population: Forty-six children (aged 3-14 years; 29 boys and 17girls) were entered into a combination L--Glu--L--Trp treatment group.The patients presented with the following diagnosed odontogenicinfections: 18 with limited purulent infection(s), 7 with focalinfection(s), 3 with diffuse infection(s). In addition, 3 patientspresented with destructive osteomyelitis and 15 with plegmons mandible.

Fifteen children, with similar demographics and infectious odontogenicdiseases, were entered into a control group treated with conventionalantibiotic therapy.

Treatment Protocol: L--Glu--L--Trp was administered im at a dosage of 2μg/kg body mass daily for 3-7 days, depending upon the severity of theinfection.

Evaluations included X-ray, clinical chemistry and serum proteintesting, and immunological testing.

Laboratory Tests: Pre-treatment measurements were performed withperipheral blood samples collected from 20 of the 46 L--Glu--L--Trptreated children to establish baseline values (Before Therapy). All 15children in the conventional treatment group were evaluated beforetherapy. Post-treatment measurements were performed with peripheralblood samples that were collected from all the conventional andL--Glu--L--Trp treated children. Fifteen healthy normal children werealso evaluated to determine the normal mean values in the respectiveassays (Normal Healthy Control). The results of these determinations aresummarized in TABLE 35.

                                      TABLE 35                                    __________________________________________________________________________    Immunology Values: Periapical Ganulomas                                                    Normal                                                                              Conventional Antibiotic                                                                     L-Glu-L-Trp Combination                                   Healthy                                                                             Before After  Before After                                 Index        Control                                                                             Therapy                                                                              Therapy                                                                              Therapy                                                                              Therapy                               __________________________________________________________________________    Leukocytes: No. (10.sup.9 /L):                                                             6.81 ± 0.26                                                                      9.20 ± 0.26†                                                               5.96 ± 0.47                                                                       10.0 ± 0.72†                                                               6.60 ± 0.39*                       % Normal Value:                                                                            (100%)                                                                              (135%) (88%)  (147%) (97%)                                 Lymphocytes: No. (10.sup.9 /L):                                                            2.62 ± 0.28                                                                      2.14 ± 0.52                                                                       1.87 ± 0.43                                                                       2.98 ± 0.30                                                                       2.37 ± 0.21                        % Normal Value:                                                                            (100%)                                                                              (82%)  (71%)  (114%) (36%)                                 T-Lymphocytes: No. (10.sup.9 /L):                                                          1.26 ± 0.13                                                                      0.63 ± 0.12†                                                               0.77 ± 0.09                                                                       0.70 ± 0.08†                                                               1.24 ± 0.11*                       % of Normal Value:                                                                         (100%)                                                                              (50%)  (61%)  (56%)  (98%)                                 B-Lymphocytes: No. (10.sup.9 /L):                                                          0.62 ± 0.11                                                                      0.49 ± 0.09                                                                       0.58 ± 0.12                                                                       0.64 ± 0.10                                                                       0.67 ± 0.05                        % of Normal Value:                                                                         (100%)                                                                              (79%)  (94%)  (113%) (108%)                                IgM (g/L)    0.98 ± 0.21                                                                      1.21 ± 0.18                                                                       1.11 ± 0.11                                                                       1.02 ± 0.08                                                                       0.93 ± 0.07                        % Normal Value:                                                                            (100%)                                                                              (123%) (113%) (105%) (95%)                                 IgG (g/L)    9.21 ± 0.16                                                                      13.06 ± 0.42†                                                              12.14 ± 0.63                                                                      12.05 ± 0.71                                                                      10.78 ± 0.83                       % Normal Value:                                                                            (100%)                                                                              (142%) (132%) (131%) (117%)                                IgA (g/L)    1.05 ± 0.11                                                                      1.91 ± 0.18                                                                       1.82 ± 0.12                                                                       1.88 ± 0.05                                                                       1.09 ± 0.04                        % Normal Value:                                                                            (100%)                                                                              (182%) (173%) (179%) (104%)                                LMIR Response: % W/ PHA:                                                                   11.60 ± 2.20                                                                     34.20 ± 2.42†                                                              24.27 ± 2.89                                                                      33.2 ± 3.23†                                                               14.65 ± 3.11*                      __________________________________________________________________________     †, p <0.05 was recorded in these studies after mathematical            comparisons of Normal values with the Pretreatment values;                    *, p <0.05 was recorded in these studies after mathematical comparisons o     Pretreatment values with the Posttreatment values.                       

The results in TABLE 35, show that prior to treatment the patients inthe trial population exhibited a statistically significant elevation intotal peripheral blood leukocytes and a decrease in T-lymphocytes (i.e.,50% and 56% of normal) but not in B-lymphocytes. FollowingL--Glu--L--Trp combination therapy, T-lymphocyte counts were increasedto 98% of normal levels, while conventional therapy only increased theT-cell counts to about 61% of normal. Leukocyte counts decreased withboth the conventional and L--Glu--L--Trp treatments.

EXAMPLE 10 Lymphatic Infections

L--Glu--L--Trp administered at a dosage of 100 μg im, or 1 μg/kgintranasally, or injected intralymphatically, is expected to control theprogression of lymphangitis.

EXAMPLE 11 Ear/Eye/Nose and Throat Infections

Protocol A: Ophthalmic Diseases

Summary Overview: Forty one patients (41; 17 men and 24 women; rangingin age from 17 to 68 years) with various eye diseases of infectious andnon-infectious origin were treated with L--Glu--L--Trp. Patients havingthe following infectious, and non-infectious, ophthalmic diseases wereentered into the trial: a) nonviral conjunctivitis, keratitis, chronicuveitis, persistent sties, and, focal chorioretinitis; and, b) retinalpigment dystrophy, Grenblad-Stranberg syndrome, and maculophathy,respectively. Five patients were Class II invalids due to visionimpairment, 4 were Class III, and 32 were impaired but not invalid.L--Glu--L--Trp was administered as an adjunct to ongoing conventionaltreatment with antibiotics, eye drops, anti-inflammatory agents, and thelike. L--Glu--L--Trp was administered daily in a single 0.1 mL (10 μg)dose by the parabulbar route (Group 1; n=21), or alternatively one dropof the dipeptide solution (5 μg) was delivered into the conjunctivalcavity twice daily on each of 5 consecutive days (Group 2; n=20). Thirtysix patients undergoing conventional therapy served as a control group(Group 3; n=36).

Clinical diagnoses of patients entered into Group 1 were as follows: 4subjects had diagnosed adenoviral conjunctivitis; 2 subjects, recurringeyelid sties; 3 subjects, chronic serous-plastic uveitis; 3 subjects,central chorioretinitis; 5 subjects, central retinal dystrophy in botheyes; 4 subjects, pigment degeneration of the retina.

Clinical diagnoses of patients entered into Group 2 were as follows:namely, 7 subjects had diagnosed chronic serous-plastic uveitis, and 13subjects adenoviral conjunctivitis.

Vision was assessed using standard measurements: i.e., field of vision,visual acuity, electrophysiological indices. Ophthalmascopic examinationwas used to evaluate and score corneal epithelial integrity, extent ofedema and infiltration of the cornea (all strata), uveitis, retinalinflammation (edema, focal and peripheral changes, exudation,hemorrhage, plasmohemorrhagia), and expansion in the vitreous body.Immune parameters in peripheral blood were also assessed. Followingtreatment, the trial subjects were followed for not less than 12 months.

Laboratory Effects: Treatment with L--Glu--L--Trp induced an increase inthe number of circulating peripheral blood B- and T-lymphocytes, and inthe numbers of CD4⁺ - and CD8⁺ -lymphocytes. Lymphokine production(i.e., measured by LMIR in response to Con-A stimulation) was increasedin all patients in Group 1 and 2 immediately following the 5 days ofL--Glu--L--Trp treatment.

Clinical Effects: Treatment with L--Glu--L--Trp resulted in a more rapidarresting of the inflammatory process and the increase in visual acuity,with a decrease being observed in the time over which medical treatmentwas required. No unfavorable response to therapy was observed.

Group 1: A "marked favorable response" to L--Glu--L--Trp therapy wasobserved (as described further below) in 9 patients (4 men; 5 women;19-52 years of age) having the following diagnosed infectious eyediseases: adenoviral keratoconjunctivitis and conjunctivitis (n=3),chronic serous-plastic uveitis (n=3), and central chorioretinitisunresponsive to conventional treatment (n=3). A "good response" toL--Glu--L--Trp therapy was observed (as described further below) in 9patients (4 men; 5 women; 17-68 years of age) having the followingdiagnosed infectious eye diseases: recurring eyelid sties (n=2);adenoviral conjunctivitis (n=1); pigment degeneration of the retina(n=2; Class II invalid vision disability); central retinal dystrophy(n=8; including 2 Class II and 1 Class III invalids for visiondisability).

Group 2: A "marked favorable response" to L--Glu--L--Trp therapy wasobserved (as described further below) in 18 patients (8 men; 10 women;20-70 years of age) having the following diagnosed eye diseases: namely,chronic serous-plastic uveitis (n=6 including 1 invalid), and adenoviralconjunctivitis (n=12). A "good response" to L--Glu--L--Trp therapy wasobserved (as described further below) in 2 patients (1 man; 1 woman; 17and 50 years of age, respectively) having the following diagnosedinfectious eye diseases: namely, serous-plastic chronic uveitis (n=1)and adenovirus conjunctivitis (n=1). A "poor response" to L--Glu--L--Trptherapy was observed in 3 patients with infectious eye diseases (2 men;1 woman; 37-49 years of age) having the following diagnosed infectiouseye diseases: central retinal dystrophy (n=1); pigment degeneration inthe retina (n=2; including a class II and a class III invalid for visionimpairment).

Group 3: As expected, since patients were selected based on theirfailure to respond to conventional therapy, response to conventionaltherapy varied between poor and nonexistent.

"Marked responses" to therapy with L--Glu--L--Trp in Groups 1 and 2 weremanifested clinically in the following objective manner: patients withadenoviral conjunctivitis recovered faster (i.e., 10-13 days) thansubjects in the control group. Patients with chronic serous-plasticuveitis experienced: i) disappearance of new eruptions; ii) completeresolution of established foci; iii) a rise in visual acuity by >0.1;and, iv) no recurrence of disease activity in the >12 month follow-upperiod. Patients with central chorioretinitis (previously nonresponsiveto conventional therapy) showed i) an increase in visual acuity; ii)increase in visual field and particularly acuity at the peripheralborders of the field; iii) disappearance of hemorrhagic foci, edema, andother manifestations of inflammation in the fundus; iv) improvements ineletrophysiological properties of the retina, i.e., values recorded inthe electroophthalmogram (EOG) recording approached normal values andSTK values also increased. "Good responses" to therapy withL--Glu--L--Trp in Groups 1 and 2 were manifested clinically in thefollowing objective manner: all patients with recurring eyelid stieswere disease free up to 6 months. Patients with adenoviralconjunctivitis were disease free for up to 6 months, recurring diseaseactivity was observed thereafter, but the relapse was shorter and milderthan previous episodes in the patient. Patients with pigmentdegeneration of the retina and central retinal dystrophy exhibited: i)an increase in visual acuity in the range of 0.05 to 0.09; ii) abroadening of the peripheral visual field borders by up to 10⁰meridians; iii) a decrease in the size of central scotomas; and, iv)improvements in electrophysiological indices (i.e., EOG values).

"Poor responses" to therapy with L--Glu--L--Trp in Group 1 weremanifested clinically in the following objective manner: patients withpigment degeneration of the retina and central retinal dystrophyexhibited i) a change in visual acuity of less than 0.05; ii) aninsignificant change in visual field; and, iii) no marked change in EOGvalues.

Overall, a marked positive patient response was observed in 75% ofpatients with infectious eye diseases (e.g., adenoviralkeratoconjunctivitis, recurring eyelid sties, recurring serous-plasticuveitis, and recurring chorioretinitis) following parabulbarintroduction of L--Glu--L--Trp; and in 90% of the patients receivingL--Glu--L--Trp by instillation. A good response was obtained in 15% ofpatients following parabulbar introduction and 10% receivingL--Glu--L--Trp by instillation. In addition to the recorded changes ininfectious and chronic diseases, L--Glu--L--Trp therapy reduced theincidence of disease recurrence and of complications. L--Glu--L--Trptreatment was found effective in 66.7% of the subjects in this trialwith infectious eye diseases.

Protocol B: Ear Diseases

L--Glu--L--Trp administered im or by the intranasal route can be used asan adjunctive therapy accompanying conventional antibiotic therapy. Thelatter route of L--Glu--L--Trp combination therapy will result inaccelerated healing of chronic and acute ear infections.

Protocol C: Corneal Diseases

L--Glu--L--Trp administered im, intranasally, or intraocularly willstimulate regeneration of corneal epithelium and restoration of visualacuity with fewer infections and complications (e.g., scarring) thanconventional therapy.

EXAMPLE 12 Occupational Radiation Exposure

Protocol A: Exposure to 100-200 R

Summary Overview: A total of 263 patients and 18 control patientssustained exposure to 100-200 Roentgens of occupational radiation over aperiod of several weeks. L--Glu--L--Trp was administered im at a dosageof 100 kg daily (or intranasally at 1 μg/kg), for 10 days. Repeatedcourses of therapy were prescribed (about every 4 to 6 months) for allpatients who exhibited periodic decreases in immunological indices.Following each treatment, L--Glu--L--Trp induced a restoration ofnormal, or near normal, peripheral blood immune indices in all patientsand functional lymphocyte activity. Clinically, treatment resulted in anarrest of asthenic syndrome, an arrest of the somatic pathologicalexacerbations, and a reduction in opportunistic infections.

Protocol B: Naval Specialists

A clinical trial was conducted of 152 naval specialists (aged 20-40years) with evidence of occupational immunologic impairments resultingfrom their exposure to radiation and occupational toxins in theKosomolets nuclear submarine sinking off the coast of Finland.L--Glu--L--Trp was evaluated for its possible effects on cellular and/orhumoral immunity. Approximately 87% of the servicemen entered into thetrial had moderate to severely impaired T-lymphocyte functionalactivity, (i.e., evidenced by decreased LMIR and blastogenesis withCon-A) and reduced neutrophil phagocytic activity (i.e., 30-40% ofnormal). Immune parameters were determined before and after a routine21-24 day rest period, during a normal rest/recuperation and trainingrotation. Servicemen were divided into two groups, the first (control)group received no treatment, and the second (experimental) group of 88servicemen received three consecutive daily intranasal doses of 100 mcgL--Glu--L--Trp.

L--Glu--L--Trp induced a pronounced increase in i) T-lymphocyte function(i.e., measured by LMIR and blastogenic response to mitogens); ii) theT-helper/T-suppressor ratio (i.e., T4/T8), which was normalized; andiii) the granulocyte lysosomal cation proteins levels, which were alsonormalized. Complement C3 levels were also returned to within the normalrange. There were no observed side-effects or indications of anyintolerance.

EXAMPLE 13 Opportunistic Infections in Transplantation

Summary Overview: L--Glu--L--Trp was administered to 17 patientsreceiving allogeneic skin grafts. 27 patients receiving similar graftsand conventional care served as the control group. L--Glu--L--Trp wasadministered im as a single daily injection of 50-100 μg on each of 5consecutive days, or alternatively, intranasally at a dose of 1 μg/kgdaily for 5 days. Graft rejection was manifest in 8 of the controlpatients. L--Glu--L--Trp treatment prevented infectious complicationsand delayed graft rejection.

EXAMPLE 14 Allergies

Protocol A: Hayfever

Summary Overview: Twenty nine patients (29) with various diagnosedallergies were treated on a daily basis for 5-7 days with L--Glu--L--Trpat a dose of 1 μg/kg delivered im or by the intranasal route. Seventeenpatients (17) in the control group received conventional therapy.Treatment with L--Glu--L--Trp resulted in disappearance of allergicreactions.

Protocol B: Drug Allergies

Summary Overview: L--Glu--L--Trp was administered to 76 patients havingclinical histories of allergies to antibiotics, during the period ofadministration of the antibiotic. A control group consisted of 43patients with similar antibiotic drug allergies. L--Glu--L--Trp wasadministered im on a daily basis for 5-10 days at a dosage of 100 μg, oralternatively, intranasally at a dosage of 1 μg/kg over the same 5-10day period. L--Glu--L--Trp treatment prevented development of allergicreactions in the majority (i.e., 70%) of the patients, and in theremaining patients, the allergic disease course was less severe.Allergic reactions were pronounced in the control group with signs ofdrug intolerance.

EXAMPLE 15 Transfusion Reactions

Summary Overview: Seventy-six patients (76) requiring hemotransfusiontherapy with allogeneic blood were treated with L--Glu--L--Trp in thepost-operative period, starting at day 4-6 post-op. Seventy-two patients(72) in a control group received conventional post-operative treatment.L--Glu--L--Trp treatment was administered as a single daily im dose of100 μg on each of 5 consecutive days, or alternatively, at a dose of 1μg/kg delivered on the same schedule by the intranasal route. None ofL--Glu--L--Trp treated patients showed clinical manifestations ofallogeneic hemotransfusion reactions. In contrast, adverse reactionswere observed in 17% of the control group.

EXAMPLE 16 Orthopedic Diseases

Protocol A: Fractures

Summary Overview: L--Glu--L--Trp was applied to 44 patients with bonefractures of various origin. The control group comprised 28 patients.L--Glu--L--Trp was administered intramuscularly or intranasally in asingle dose of 100 μg daily for 10 days. The use of L--Glu--L--Trpaccelerated essentially (in comparison with the control group) theconsolidation of fractures, prevented the development of infectiouscomplications, reduced pain syndrome and treatment duration.

Protocol B: Chronic Osteomyelitis

Summary Overview: L--Glu--L--Trp was administered to 176 patients withchronic osteomyelitis of various etiology and bone locations. Thecontrol group consisted of 88 patients receiving conventional treatment.L--Glu--L--Trp was administered im as a single daily dosage of 100 μg,or intranasally at a dose of 1 μg/kg daily, over a period of 10 days.L--Glu--L--Trp treatment resulted in a pronounced positive effect onclinical course that was expressed as a significant decrease in systemictoxicity and pain, disappearance of purulent inflammation, acceleratedwound healing, decreased size of the areas of bone destruction, anddecreased incidence of clinical relapse.

Protocol C: Acute Osteomyelitis

Trial Population: Seventeen hospitalized children (10 boys and 7 girls)were entered into this protocol: 6 with acute inflammatory diseases ofsoft tissues (abscesses, phlegmons), 4 with odotogenous osteomyelitis ofthe jaw, and 7 with hematogenous osteomyelitis of the flat and tubularbones. In 11 children, the course of the illness was acute and in 6 itwas subacute and chronic.

Treatment Protocol: L--Glu--L--Trp was administered in combinationtherapy with conventional antibiotic therapies. Where necessary, surgerywas performed to drain infectious foci in the jaw or soft tissues,affected teeth were extracted, and for osteomyelitis a port was drilledfor infusion of antibiotics. Physical examination and laboratory testingwas conducted before administering L--Glu--L--Trp and 2-3 days after thetreatment.

Laboratory tests were conducted to determine clinical chemistry andprotein values prior to and after therapy (i.e., including theconcentrations of bilirubin, creatinine, albumin, cholesterol, ureanitrogen, glucose, protein, chloride; potassium, phosphorus, calcium,sodium, alkaline phosphatase, α₁ -orosomucoid, α₂ -macroglobulin,prealbumin, ceruloplasmin, and transferrin). While urea nitrogen andcholesterol decreased slightly after therapy, these and all otherchanges observed after L--Glu--L--Trp treatment were not statisticallysignificant. The results of differential cell counts and testing ofimmune parameters are summarized in TABLE 36.

                                      TABLE 36                                    __________________________________________________________________________    Immunology Values                                                                            After                 After                                             Before                                                                              L-Glu-L-Trp     Before                                                                              L-Glu-L-Trp                              Index    Therapy                                                                             Therapy                                                                             Index     Therapy                                                                             Therapy                                  __________________________________________________________________________    Leukocytes: (10.sup.9 /L):                                                             8.7 ± 0.96                                                                       7.0 ± 0.4†                                                                Lymphocytes:                                                                  % of PBL: 29 ± 3.7                                                                         43 ± 3.61†                                          No. (10.sup.9 /L):                                                                      2.3 ± 0.33                                                                       3.15 ± 0.41                           B-Lymphocytes:       T-Lymphocytes:                                           % of PBL:                                                                              9.5 ± 1.24                                                                       15.1 ± 3.32†                                                              % of Lym: 59.6 ± 5.12                                                                      66.2 ± 2.57                           No. (10.sup.9 /L):                                                                     0.29 ± 0.11                                                                      0.41 ± 0.11                                                                      No. (10.sup.9 /L):                                                                      1.6 ± 0.36                                                                       1.7 ± 0.3                             Null-Lymphocytes:    T-helper                                                 % of PBL:                                                                              30.8 ± 4.94                                                                      18.7 ± 4.68†                                                              (E.sub.tr -RFC):                                                                        50 ± 6.71                                                                        57.3 ± 6.34                           No. (10.sup.9 /L):                                                                     0.8 ± 0.18                                                                       0.42 ± 0.11†                                                              % of Lym: 1.2 ± 0.3                                                                        1.4 ± 0.24                                                 No. (10.sup.9 /L):                                       Neutrophils:         T-suppressor:                                            % PBL:   63 ± 4.63                                                                        50 ± 4.8†                                                                 % of Lym: 9.5 ± 3.18                                                                       8.8 ± 5.4                             × 10.sup.9 /L                                                                    5.8 ± 0.93                                                                       3.4 ± 0.33†                                                               No. (10.sup.9 /L):                                                                      0.26 ± 0.09                                                                      0.3 ± 0.2                             LMIR: % PHA:                                                                           93.7 ± 29.2                                                                      47.7 ± 11.2†                                                              LMIR: % Con A                                                                           79 ± 5.85                                                                        49.7 ± 13.7†                   IgM (g/L)                                                                              2.19 ± 0.65                                                                      1.64 ± 0.26†                                                              IgG (g/L) 14.3 ± 2.42                                                                      14.4 ± 1.31                           IgA (g/L)                                                                              1.63 ± 0.2                                                                       1.68 ± 0.18                                                                      C3 (g/L)  0.82 ± 0.03                                                                      0.72 ± 0.05                           __________________________________________________________________________     †, a p value of <0.05 was recorded in these studies after              mathematical comparisons of Pretreatment and Posttreatment values.       

The results presented in TABLE 36 show that prior to treatment thepatients in the trial population had decreased levels of B- andT-lymphocyte mitogen responsiveness to PHA and Con-A, (i.e, as measuredby cytokine production in LMIR assay). Prior to treatment, the patientsalso showed possible decreases in the percentages of T-helperlymphocytes and increases in T-suppressor lymphocytes, i.e., asdetermined using a trypsin-treated erythrocyte rosette forming cell(E_(tr) -RFC) assay, wherein T-helper cells are trypsin resistant(E_(tr) -RFC) and T-suppressor cells are trypsin sensitive (E_(ts)-RFC). Following treatment with L--Glu--L--Trp the following changeswere observed: i) leukocyte counts and neutrophil counts droppedsignificantly; ii) Null lymphocyte counts dropped; and, iii)B-lymphocyte counts and serum IgM levels dropped significantly. Whilenot statistically significant, lymphocyte counts in peripheral bloodincreased with T-helper rising and T-suppressor percent falling.T-lymphocyte function increased as measured by cytokine production invitro in the LMIR assay in response to PHA and Con-A mitogens andstaphylococcal and streptococcal antigens.

Clinical Response: Children in the treatment group reported feelingbetter, and according to the report of the attending physician "woundscleaned themselves of necrotic tissue and developed granulation tissue2-3 days earlier than usual."

Protocol D: Acute Osteomyelitis

In 29 adolescents with osteomyelitis, 2 μg/kg L--Glu--L--Trp wasadministered im daily on each of 3-7 consecutive days, depending uponthe severity of the infection. The control group consisted of 13children who received conventional therapy with antibiotics. Clinicalimprovement was noted in the 3 days following L--Glu--L--Trp treatmentwith an arrest in the inflammatory process, a reduction in pain, and anincreased stability of the underlying dental structures as evidenced byX-ray studies. Fevers reportedly resolved on the average 1-2 days soonerin the L--Glu--L--Trp treated group, and all wounds and purulentdischarge were observed to close with healing 2-3 days earlier thanchildren the control group. In only 1 child (i.e., having purulentosteomyelitis), out of the 29 L--Glu--L--Trp-treated, did the woundsfailed to heal (i.e., a 3% failure rate), as compared with failure in 2of the 13 children in the control group (i.e., a 15% failure rate).Clinical improvements in the L--Glu--L--Trp treated children wereaccompanied by decreases in the total leukocyte counts, and the T-cellcounts nearly doubled.

EXAMPLE 17 Kidney and Prostate Diseases

Protocol A Pyelonephritis in Pregnancy

Summary Overview: Twenty-seven pregnant female patients (27) withpyelonephritis were treated with L--Glu--L--Trp as a single daily doseof 100 μg on each of 5-10 consecutive days and in combination withongoing conventional therapy. A control group was constituted of 19control patients with pyelonephritis receiving the conventional therapy.Treatment with L--Glu--L--Trp resulted in reduction of fever, thenormalization of urinary output, and overall clinical improvement withresolution of infection. Women treated with L--Glu--L--Trp experiencednormal delivery without complications.

Protocol B Prostatitis

Trial Population: Thirty four patients (aged 22 to 45 years) withchronic prostatitis were entered into Group 1 of this trial during theacute phase of their disease. Diagnosis was based on medical history,and physical examination of the prostate (palpated through the rectum).Laboratory testing included microscopic examination of prostate glandsecretions, spermography and urinalysis. All patients had previouslyreceived full courses of unsuccessful therapy with antibacterial agents,and additional individualized therapy including uroantiseptics,spasmolytic, ganglion blockers, novocain paraprostate and presacralblocks, ultrasound, and prostate gland massage. Success of anyindividualized therapy was only partial and short-term.

A control group (Group 2) constituted 14 patients (aged 23-45), alsohaving chronic prostatitis, and enrolled during the active phase of thepatients' disease. All patients in the control group had also failedpreviously one or more full courses of antibiotic and conventionaltherapy.

Treatment Protocol: L--Glu--L--Trp was administered im daily at 100μg/dose for 5 days (500 μg total treatment course) in combination withconventional individualized therapy including antibacterial preparationsand uroantiseptics (e.g., oletrin, and bisepton), physical procedures(e.g., ultrasound; using a Sterzhen 1 instrument), prostate glandmassage, and exercise

Patients in Group 2 received only conventional therapy withantibacterial agents and individualized treatments, (i.e., as above).

Laboratory Tests: Peripheral blood samples were collected for laboratorytesting 10 days after the last injection of L--Glu--L--Trp. The resultsof the laboratory testing for immune parameters are presented in TABLE37.

                                      TABLE 37                                    __________________________________________________________________________    Immunology Values (X ± S.D.)                                                           Normal ± SD                                                                       Conventional                                                                              L-Glu-L-Trp                                    Index       or Range                                                                             Before                                                                              After Before                                                                              After                                    __________________________________________________________________________    T-Lymphocytes: % of Lym:                                                                  54.0 ± 2.1                                                                        43.4 ± 4.4                                                                       41.7 ± 5.8                                                                       45.8 ± 2.3                                                                       44.4 ± 2.3                            No. (10.sup.9 /L):                                                                        --     0.96 ± 0.01                                                                      0.92 ± 0.03                                                                      0.91 ± 0.02                                                                      0.88 ± 0.1                            B-Lymphocytes: % of PBL:                                                                  24.0 ± 0.6                                                                        23.2 ± 2.3                                                                       24.8 ± 2.3                                                                       24.3 ± 1.5                                                                       27.2 ± 1.4                            No. (10.sup.9 /L):                                                                        --     0.46 ± 0.03                                                                      0.5 ± 0.1                                                                        0.48 ± 0.1                                                                       0.54 ± 0.1                            Phagocytes: % PBL:                                                                        23.3 ± 0.4                                                                        32.4 ± 2.6                                                                       29.7 ± 2.7                                                                       41.1 ± 3.8                                                                       39.4 ± 3.5                            Complement(CH.sub.50)                                                                     30.3 ± 0.2                                                                        29.2 ± 1.1                                                                       28.5 ± 1.1                                                                       29.5 ± 0.6                                                                       29.4 ± 0.5                            Immune Complexes (units)                                                                  0.06-0.08                                                                            0.09 ± 0.01                                                                      0.08 ± 0.01                                                                      0.86 ± 0.01                                                                      0.80 ± 0.004                          IgM(g/L) % Normal Value:                                                                  0.6-3.8                                                                              2.1 ± 0.9                                                                        2.3 ± 4.1                                                                        2.1 ± 0.2                                                                        2.1 ± 0.1                             IgG(g/L) % Normal Value:                                                                  6-18   12.8 ± 1.2                                                                       12.5 ± 1.3                                                                       12.6 ± 0.2                                                                       12.5 ± 0.6                            IgA(g/L) % Normal Value:                                                                  0.8-5.2                                                                              2.5 ± 0.3                                                                        2.4 ± 0.2                                                                        2.5 ± 0.2                                                                        2.2 ± 0.1                             __________________________________________________________________________     †, a p value of <0.05 was recorded in these studies after              mathematical comparisons of Pretreatment and Posttreatment values.       

Considering that not all of the subjects receiving L--Glu--L--Trp(Group 1) responded to therapy with a change in immune parameters, thedata were evaluated in an attempt to identify individual subgroups ofpatients responsive to therapy. Patients were grouped according towhether the pre-treatment immune parameter was i) normal, ii) decreased,or iii) increased (relative to the normal healthy range of values). Theresults of these analyses are presented in TABLE 38.

                                      TABLE 38                                    __________________________________________________________________________    Immunology Values: Grouped by Putative Immune Parameter Defect (X ±        S.D.)                                                                         L-Glu-L-Trp            L-Glu-L-Trp                                            Index      Before                                                                              After Index    Before                                                                              After                                   __________________________________________________________________________    T-Lymphocytes (%):     Imm. Cmplx. (units)                                    Normal (n = 18):                                                                         54.0 ± 2.1                                                                       50.0 ± 3.6                                                                       Normal (n = 7):                                                                        0.07 ± 0.03                                                                      0.08 ± 0.01                          Decreased (n = 14):                                                                      31.1 ± 1.0                                                                       37.9 ± 2.2                                                                       Decreased (n = 8):                                                                     0.05 ± 0.001                                                                     0.07 ± 0.01                          Increased (n = 2):                                                                       74.5 ± 2.1                                                                       40.0 ± 15.5                                                                      Increased (n = 19):                                                                    0.11 ± 0.01                                                                      0.08 ± 0.01†                  B-Lymphocytes:         IgM (g/L)                                              Normal (n = 16):                                                                         24.0 ± 0.6                                                                       29.2 ± 2.2                                                                       Normal (n = 33):                                                                       2.01 ± 0.13                                                                      2.08 ± 0.12                          Decreased (n = 10):                                                                      15.0 ± 1.2                                                                       26.0 ± 2.9†                                                               Increased (n = 1):                                                                     6.0   3.44                                    Increased (n = 8):                                                                       36.5 ± 2.2                                                                       24.8 ± 2.9†                                        Phagocytes (% of PBL): IgG (g/L)                                              Normal (n = 3):                                                                          23.3 ± 0.4                                                                       58.6 ± 18                                                                        Normal (n = 26):                                                                       11.8 ± 0.6                                                                       12.5 ± 0.7                           Decreased (n = 5):                                                                       16.6 ± 4.2                                                                       36.6 ± 8.5†                                                               Decreased (n = 2):                                                                     3.7 ± 0.4                                                                        7.8 ± 1.1†                    Increased (n = 26):                                                                      47.9 ± 4                                                                         37.7 ± 3.9                                                                       Increased (n = 6):                                                                     19.1 ± 0.3                                                                       14.7 ± 1.4                           Complement (CH.sub.50) IaG (g/L)                                              Normal (n = 13):                                                                         30.3 ± 0.2                                                                       29.3 ± 0.9                                                                       Normal(n = 34):                                                                        2.50 ± 0.15                                                                      2.20 ± 0.12                          Decreased (n = 14):                                                                      26.3 ± 0.5                                                                       34.2 ± 0.9†                                        Increased (n = 7):                                                                       34.2 ± 0.9                                                                       30.7 ± 0.9                                                __________________________________________________________________________     †, p < 0.05 statistical difference between Pre and Posttreatment       values.                                                                  

The results presented in TABLE 38, above, show that treatment of thepatients in Group 1 with L--Glu--L--Trp i) did not significantly alterimmune values in patients that had "normal" pre-treatment values; ii)significantly increased B-lymphocytes, phagocytes, and complement andIgG levels in patients that had "decreased" pre-treatment values; and,iii) significantly decreased B-lymphocytes and immune complex levels inpatients that had "increased" pre-treatment values. The same analysiswas conducted using the data collected following conventional therapy ofthe patients in Group 2, and none of the differences were statisticallysignificant.

Clinical Results: Clinical results were recorded according to acceptedcriteria for evaluating the efficacy of treatments for prostatitis:namely, i) disappearance of painful symptoms over a period of 1-1.5months (e.g., absence of pain in perineum, sacrum, and scrotum); ii)size and elasticity of the prostate on physical examination; iii) volumeof prostate gland secretions and ejaculate; iv) microscopic features ofsecretions and ejaculate (e.g., leukocytes, macrophages, phagocytelecithin granules, spermatozoids, squamous epithelial cells,erythrocytes), v) volume of urine; and, vi) overall patient health. A"good" result was scored if laboratory values were normalized and thepatient remained pain free during the 1-1.5 month follow-up period ofexamination. A "satisfactory" result was scored if laboratory valueswere normalized even if some pain remained. An "unsatisfactory" resultwas scored if laboratory values remained unchanged and pain persisted.

A "good" result was recorded in 26 patients (76.5%) in Group 1 followingL--Glu--L--Trp treatment. After finishing the treatment course, patientsnoticed significant improvement in general physical condition withdisappearance of painful symptoms and dysuria. All the laboratoryindicia returned to within the normal range of values. A "satisfactory"result was recorded in the remaining 8 patients in Group 1 (23.5%). No"unsatisfactory" results were recorded with the patients in Group 1.

In contrast, following conventional treatment, the patient responses inGroup 2 were scored as follows: 9 "good" (64.3%); 3 satisfactory(21.4%); and, 2 (14.3%) unsatisfactory

EXAMPLE 18 Clinical Studies: Leprosy

Overview: A total of 45 patients with leprosy (Hansen's disease) weretreated with L--Glu--L--Trp administered im daily at a dose of 100 μg,or intranasally daily at a dose of 1 μg/kg, on each of 5 consecutivedays as an adjunct to ongoing conventional therapy. Twenty-seven otherM. leprae infected individuals constituted the control group. Thepatients studied had previously documented antibiotic resistance totreatment by conventional methods. L--Glu--L--Trp treatment resulted ini) resolution of dermal leprotic lesions, ii) lower incidence ofrecurrence of dermal disease, and iii) accelerated healing of individualdermal ulcers.

Infection with M. leprae is well recognized to result inimmunosuppression. The immune indices in the peripheral blood ofL--Glu--L--Trp treated patients were normalized, and certain patientsacquired skin test responsiveness to lepromin antigen.

Background: Infection with Mycobacterium leprae generally proceeds in aslow inexorable and progressively debilitating manner through stages oftuberculoid leprosy to lepromatous leprosy and eventual death. Thedisease progression is gradual from non-differentiated leprosy (J) toborderline tuberculoid (BT) to polar tuberculoid (TT) to borderline (BB)to borderline lepromatous (BL) to polar lepromatous (Llp). Thelepromatous stage of the disease is further subdivided into subpolarlepromatous (Lls). Histological features of acid fast bacteria intissues, perivascular granulomas in skin and mucous membranes are wellestablished, as are the general immune suppression evident as theabsence of a delayed-type hypersensitivity skin reaction to leprominantigen, decreased blast transformation of peripheral blood lymphocytesto PHA, an altered ratio of CD4⁺ /CD8⁺ lymphocytes in peripheral blood,and decreased levels of IgM and IgG in serum. The therapy of choice mostcommonly employs several anti-leprotic preparations inoptimally-tolerated doses, e.g., diaminodiphenylsulfone (DDS) takeninternally at a dose of about 50 mg and sodium sulfone takenintramuscularly using a 50% sterile injection solution. The minimal timeframe for therapy of TT leprosy is 3 years; for non-differentiatedleprosy 5-7 years; and, for LL leprosy, patients must receive treatmentover their entire lives. Treatment courses are commonly 6 months with anintermission of 1-1.5 months between courses, and to prevent thedevelopment of drug resistant bacteria, the drug preparation ispreferably changed to a new drug (e.g., Rifampicin, ethionamide,prothionamide or Lampren-clofaximine) after each course of therapy.

Trial Protocol: An assessment was made of disease progression prior toand after combination therapy involving physical examination of theskin, biochemical testing of blood and urine, bacteriologic testing ofskin plaques, immunological tests of cellular and humoral immunity, andhistologic and electron microscopic examination of cutaneous skinlesions.

Patient Population: Efficacy of L--Glu--L--Trp in combined antibiotictherapy was evaluated in 45 patients (aged 18 to 83; 23 men and 22women) with chronic debilitating infection with Mycobacterium leprae,i.e., Hansen's disease (the length of illness varied from 2 to 25years). According to standard diagnostic criteria at clinicalpresentation: 18 patients had active leprosy; and in 27 the disease wasregressive. Sixteen of the patients with active leprosy also presentedwith neurotrophic ulcers (NTU) and chronic osteomyelitis. According tothe Ridley-Jopling diagnostic classification criteria, 37 patients weresuffering from lepromatous leprosy, 6 from borderline-lepromatousleprosy, and 2 from borderline tuberculous leprosy. The length ofdisease in this patient population was 2 to 25 years. None of thepatients in the trial had received previous treatment with animmunomodulator, and all were receiving conventional courses ofsulfone-type antibiotic therapy and experiencing prolonged slow-diseaseprogression.

L-Glu-L-Tip Treatment: The 18 patients with active leprosy were treatedwith L--Glu--L--Trp administered im at a dosage of 1 μg once daily inabout 1 mL for 5 days. In 10 of the patients with the regressive courseof disease, the L--Glu--L--Trp was administered daily intranasally bydrops at a dosage of 1 μg daily for 5 days (i.e., one treatment course).Four of the 16 patients with active leprosy and NTU were treated withadditional localized intra-ulcer injections of about 1-20 μg/siteL--Glu--L--Trp. While L--Glu--L--Trp was being administered, antibiotictherapy was discontinued.

Summary of Immunological Studies: Parameters of cellular and humoralimmunity were tested 5-7 days after concluding one treatment course inthe 16 patients with active leprosy. Nonspecific immunosuppression of M.leprae infection was evident in the patient population prior toinitiating therapy with only about 13-15% of patient lymphocytes capableof mitogen-induced blast transformation (TABLE 38). This contrasted with40-60% blast cells in normal subjects following culture with PHA(polyvalent mitogen), or 20-40% blast cells with Con-A (T-cell mitogen;TABLE 39; control). After the 5-7 day course of L--Glu--L--Trptreatment, blast responsiveness of patient lymphocytes was increased1.8-fold to PHA and 1.6-fold to Con-A, and after 6 months (and severaltreatment courses), responsiveness to Con-A was in the normal range(i.e., 20-40% of lymphocytes responsive).

                  TABLE 39                                                        ______________________________________                                        Percentage of Peripheral Blood Lymphocytes                                    Responsiveness to PHA and Con A                                               Treatment       PHA (%).sup.a                                                                             Con-A (%).sup.a                                   ______________________________________                                        Before L-Glu-L-Trp                                                                            15.2 ± 4.51                                                                            13.9 ± 2.9                                     5-7 days        128.7 ± 3.8                                                                            22.5 ± 4.0                                     6 months        122.3 ± 1.43                                                                           36.9 ± 1.28                                    None-healthy controls                                                                         40-60*      20-40*                                            ______________________________________                                         .sup.a Mean % responsive PBL (blast cells); patient values +/- S.D.           (number of patients = 16);                                                    *Normal range of values for lymphocytes from healthy control subjects (n      50).                                                                     

The percentage of antigen-specific patient lymphocytes responsive tosynthetic leprosy antigen was also increased by about 1.6-fold on day5-7 of treatment from a pre-treatment mean value of 1.78±0.7% blastcells to a value of 2.86±0.9%.

Lymphocyte sub-populations were evaluated in leprosy patients before andafter treatment and the results are presented in TABLE 40.

                                      TABLE 40                                    __________________________________________________________________________    Peripheral Blood Lymphocyte Subpopulations in Leprosy Patients                Before and After 5-7 Days Treatment with L-Glu-L-Trp*                         Patient Group                                                                          Treatment                                                                          OKT 11 (%)                                                                          OKT 8 (%)                                                                           OKT 4 (%)                                                                           OKT 4/OKT 8                                   __________________________________________________________________________    Active Disease                                                                         Before                                                                             72.13 ± 3.34                                                                     26.00 ± 1.49                                                                     45.71 ± 4.99                                                                     1.83 ± 0.26                                         After                                                                              72.22 ± 1.52                                                                     25.56 ± 1.15                                                                     40.67 ± 1.44                                                                     1.61 ± 0.08                                Regressive Disease                                                                     Before                                                                             76.25 ± 3/82                                                                     27.75 ± 2.69                                                                     37.00 ± 4.09                                                                     1.33 ± 0.05                                         After                                                                              76.25 ± 0.87                                                                     27.25 ± 3.42                                                                     43.25 ± 1.91                                                                     1.68 ± 0.32                                Health controls**                                                                      None 72.5 ± 1.42                                                                      23.55 ± 1.24                                                                     44.55 ± 1.67                                                                     1.88 ± 0.09                                __________________________________________________________________________     *Mean patient values % +/- S.D. (number of patients = 16);                    **Normal percentage for lymphocytes from healthy control subjects (n =        50).                                                                     

The results in TABLE 40 show that the total percentage of T-lymphocytes(OKT 11⁺) in peripheral blood was not significantly altered aftertherapy; the percentage of OKT 4⁺ T-helper cells in circulation inpatients with active disease was slightly decreased (possibly byredistribution into infected tissues); and, in patients with regressivedisease, the percentage of OKT 4⁺ cells in circulation was slightlyincreased. The observed increase in mitogen-induced blast responsivenessof lymphocytes in the leprosy patients after 5-7 days of L--Glu--L--Trptreatment was accomplished without a major bulk change in either thenumber or subpopulation composition of peripheral blood T-lymphocytes.

Summary of Histological Findings: Three indices were used to scoreserial sections of biopsy samples obtained from patients before andafter treatment with L--Glu--L--Trp, namely, i) bacterial index (BI),i.e., the relative saturation of the tissue with mycobacterial bacilliin tissue sections on a scale of 0 (low) to 1 (high); ii) histologicindex (HI), i.e., scoring the area of granuloma and bacteria (Materialsand Methods, below); and, iii) esterase index (EI), scoring thedifferentiative state of the granuloma (Materials and Methods, below).The results of these studies with patients withregressive-differentiated lepromatous leprosy (LL_(D)),undifferentiated-active lepromatous leprosy (LL_(S)), borderlinelepromatous leprosy (BL), and borderline tuberculoid leprosy (BT) aresummarized in TABLE 41.

                                      TABLE 41                                    __________________________________________________________________________    Histologic Examination at One Month of Dermal Lesions in                      Leprous Patients Before and After L-Glu-L-Trp Treatment*.                     Disease State                                                                       Treatment                                                                           BI    HI    EI    Gross Pathology                                 __________________________________________________________________________    LL.sub.D                                                                            Before                                                                              0     1.83 ± 0.03                                                                      0.6 ± 0.01                                                                       maculation; surface infiltration                (n = 3)                       lepromas on torso and extremities                     1 Month                                                                             0     135 ± 0.03                                                                       0.91 ± 0.2                                                                       no visible change                                     After       (↓ 26%)                                                                      (↑ 52%)                                               3 Months                                                                            0     0.66 ± 0.1                                                                       1.9 ± 0.5                                                                        pronounced decrease in surface                        After       (↓ 64%)                                                                      (↑ 217%)                                                                      infiltration and macules                              6 Months                                                                            0     0.56 ± 0.05                                                                      2.15 ± 0.45                                                                      complete disappearance of surface                     After       (↓ %)                                                                        (↑ 258%)                                                                      infiltrate and partial disappearance                                          of deep infiltrate                              LL.sub.S                                                                            Before                                                                              0.97 ± 0.03                                                                      3.37 ± 0.04                                                                      0.55 ± 0.01                                                                      thickening and sensitivity of                   (n = 10)                      peripheral nerve stems                                1 Month                                                                             0.63 ± 0.03                                                                      3.24 ± 0.04                                                                      0.79 ± 0.3                                                                       decrease skin infiltration                            After (↓ 35%)                                                                            (↑ 44%)                                               3 Months                                                                            0.39 ± 0.09                                                                      0.07 ± 0.01                                                                      1.17 ± 0.6                                                                       disappearance of deep infiltrations;                  After (↓ 60%)                                                                      (↓ 70%)                                                                      (↑ 113%)                                                                      resolution of surface; regression of                                          deep lepromas                                         6 Months                                                                            0.38 ± 0.05                                                                      2.00 ± 0.02                                                                      0.55 ± 0.01                                                                      decrease in size/regression of                        After (↓ 61%)                                                                      (↓ 38.1%)                                                                          lepromas                                        BL    Before                                                                              1.04 ± 0.5                                                                       3.37 ± 0.9                                                                       0.73 ± 0.3                                         (n = 4)                       infiltration, papules on skin torso                                           and extremities                                       1 Month                                                                             0.99 ± 0.3                                                                       2.71 ± 0.1                                                                       2.03 ± 0.25                                                                      signs of regression of papules and                    After       (↓ 20%)                                                                      (↑ 178%)                                                                      decreased infiltration of skin                        3 Months                                                                            0.72 ± 0.05                                                                      0.53 ± 0.1                                                                       3.45 ± 0.3                                                                       regression of papules,                                After (↓ 31%)                                                                      (↓ 84%)                                                                      (↑ 372%)                                                                      disappearance of surface and deep                                             infiltrates                                           6 Months                                                                            0.53 ± 0.07                                                                      1.25 ± 0.1                                                                       1.37 ± 0.1                                                                       disappearance of dermal macules,                      After (↓ 46%)                                                                      (↓ 54%)                                                                      (↓ 33%)                                                                      resolution of infiltrated                       BT    Before                                                                              0     2.9   0.15  extensive plaques in the skin of                (n = 1)                       extremities; hyperemia; edema                         1 Month                                                                             0     2.05  0.51  plaques, scaling of skin, dermal                      After                   edema all disappearing                                3 Month                                                                             0     2.05  0.51  decrease in area of plaques,                          After       (↓ 29%)                                                                      (↑ 240%)                                                                      papules, and surface scaling                          6 Months                                                                            0     0     0.06  a few residual macules on the skin,                   After             (↑ 250%)                                                                      atrophy/scarring                                __________________________________________________________________________

The results presented in TABLE 41, showed decreases in the histologicalindex, i.e., decreased areas involved with bacteria and granulomatousinfiltration in the skin biopsy samples obtained from all the patientsstudied at one month post-treatment with L--Glu--L--Trp. The histologicfindings also suggested a trend toward a higher level of macrophageactivation and greater differentiated state of the existing granulomatain the tissues. The latter findings are highly suggestive of increasedbactericidal activity in the residual granulomas. Consistent with thisinterpretation was a general improvement in the gross pathology of theskin in the treated patient population when compared with thepretreatment pathology. Dramatically, in the patients with disease inregression (i.e., LL_(D)) what was before treatment a homogeneousmycobacterial infection virtually disappeared after therapy with adecrease in the area of granulomas and an increase in the number oflymphocytes, mononuclear cells, fibroblasts, mast cells, histiocytes,fibroblasts and fat cells in the skin. In patients with active disease(i.e., LL_(S)), the number of macrophages containing bacilli was greatlyreduced and the number of lymphoid cells, histiocytes and fibroblastsincreased. Histological measurements performed at 3 months werestriking, with signs of disease regression in all patients in the trialas evidenced by decreased recognizable bacilli in the tissues (i.e.,decreased BI), decreased granulomata and cellular infiltration in thetissues (i.e., decreased HI), and more activated macrophages anddifferentiated granulomata (i.e., increased EI). Histologic and electronmicroscopic examination confirmed degenerative changes in the residualbacteria in patients' tissues, as evidenced by an increase in fatcontent in the cytoplasm of the bacilli, an increase in fuchsinophilicgranules in tissues (degenerative mycobacterial bodies), and an increasein degenerative forms of leprous bacilli and cell wall materials visiblein phagolysosomes of tissue macrophages and histiocytes. Signs of tissuewould healing were also evident as neovascularization of tissues at theformer granuloma sites. By six months (the time at which potentialefficacy of a leprosy drug is normally assessed) histologic signs of agranulomatous response were waning with very few bacteria being evident,and decreased tissue infiltrate of immune cells.

The results indicated that introduction of L--Glu--L--Trp into thetreatment plan for M. leprae greatly accelerated the resolution of skinlesions in all patients in the trial group. Dramatically, four subjectswith negative skin tests for lepromin at the beginning of therapy turnedpositive. Other clinical evidences of success are disclosed below.

Summary of Clinical Results: Six to seven days after the completion of asingle course of L--Glu--L--Trp therapy, no visible changes wereclinically evident. However, after 1 month of therapy, the firstclinical effects became evident as decreased sizes of measured cutaneouslesions in the 6 patients with borderline lepromatous leprosy and the 2patients with borderline tuberculous leprosy, although the clinicaleffects varied from patient to patient. No change was noted in thepatients with progressive lepromatous leprosy, but some improvements incutaneous lesions were noted in a few of the patients with staticlepromatous leprosy.

After three months of treatment pronounced signs of dermal improvementwere clinically evident in responsive patients, i.e., lepromatouspapules, plaques, lepromas, and infiltrates were decreased.

After 6 months of treatment with L--Glu--L--Trp, cutaneous lesions werenearly completely absent from responsive patients, and the size of theremaining leprous plaques was greatly decreased in size with lucid zonesof scar tissue forming about the periphery of the plaques. In somepatients leprous plaques were completely absent and replaced by atrophicmaculas having a burgundy-like color. Remarkably, one patient previouslyclassified as borderline lepromatous leprosy was down-graded toborderline tuberculous leprosy with nearly a 100% reduction inhistological evidence of infection, and complete histologicaldisappearance of cutaneous M. leprae. A second patient, previouslyclassified with tuberculous leprosy showed a complete cessation ofleprous infection and was discharged to out-patient care.

At 6 to 12 months, two additional patients classified at the beginningof the trial with lepromatous leprosy and borderline leprosy werereleased to out-patient ambulatory care, with cutaneous and histologicalevidence of their disease completely absent. In one case a perivascularlepromatous granuloma evident by gross pathology at the beginning oftreatment was histologically examined and seen to be resolved to adiffuse lympho-histiocytic infiltrate completely devoid of mycobacteria.Histological examination of biopsy samples from skin (and nasal mucousmembranes) and taken from the locations of previous lesions all revealeda complete absence of mycobacteria or perivascular leprous granulomata,and instead, there was evidence of diffuse infiltration with smalllymphocytes and histiocytes (dendritic and Langerhans cells).

Within the treatment subgroup of 16 patients who had neurotrophic ulcersat the beginning of the trial (NTU; above), 4 patients initially hadpresented with primary disease, 9 with recurrent NTU, and 3 with chronicoscheal osteomyelitis of the lower extremities complicated with NTU. Atthe beginning of therapy, the ulcerative lesions were filled withpurulent-necrotic material and no evidence of wound healing was present.Fifteen of the 16 patients showed clinical improvements while onL--Glu--L--Trp therapy. For primary NTU, the clinical improvements wereobtained after about 3-6 weeks of L--Glu--L--Trp treatment, i.e., withsecretions from the ulcer disappearing; for recurrent NTU theimprovements were obtained at about 4-6 weeks (in both cases, theresults obtained were dependent on the size of the ulcers). The ulcerswere replaced for the most part by a dried crusty scab, and eventually acallous. At about the same time chronic osteomyelitis of the lowerextremities was healed in the three patients so afflicted. Cytologicalexamination of smears taken from the ulcers during treatment revealed adecrease in acid fast bacteria in the exudates, and after only one weekof therapy, activated lymphocytes, monocytes, and neutrophils wereobserved in the exudates (TABLE 42). Histologic evidence ofre-epithelialization of the ulcers was evident after only two weeks ofL--Glu--L--Trp therapy.

                                      TABLE 42                                    __________________________________________________________________________    Cytology of Smears from Lepromatous Ulcers in Patients                        Treated with L-Glu-L-Trp                                                            Romanovsky         Lysosome Cation                                                                       HCT-test                                     Treatment                                                                           Staining           (OD imits)                                                                            (OD units)                                   __________________________________________________________________________    Before                                                                              degenerative necrotic cells                                                                      0       0                                            Day 2 degenerative cells & inflammatory cells                                                          0.33 ± 0.02                                                                        0.09 ± 0.01                               Day 4 regenerative: 1st or 2nd phase                                                                   0.42 ± 0.05                                                                        0.11 ± 0.1                                Day 7 regenerative: 2nd or 3rd. phase, distinctive                                                     0.58 ± 0.03                                                                        0.59 ± 0.2                                      cellular elements gone from smears and                                        replaced by neutrophils                                                 Day 14                                                                              regenerative: 3rd. phase/healing; lymphoid                                                       0.84 ± 0.09                                                                        0.78 ± 0.3                                      and monocytoid cells; epithelial cells in                                     smears                                                                  __________________________________________________________________________

In treating patients with NTU, clinical improvement was noted in 15 ofthe 16 patients. In patient #16, chronic osteomyelitis was not improved.Healing times for NTU in the treated patients were about 3-6 weeks forprimary NTU (depending upon size), and about 4-6 weeks for secondaryrecurrent lesions. The measured rate of healing for primary NTU (fromplanimetry) was 6.34 mm² /day, and for recurrent NTU the rate was 5.91mm² /day. The area of ulceration decreased over the first week oftreatment by a mean value of 40% in patients with primary NTU, and by22.6% in those with recurrent ulcers. By the end of the second week theadditional decrease in area of ulceration was 22% and 29%, respectively;and, by the third week 23.2% and 8.2%.

While the trial was terminated at 6 months, at twelve months, fourpatients (3 with LL and 1 with BL) who received repeat courses ofL--Glu--L--Trp over the period from 6-12 months continued to be observedand their condition continued to improve with further regression of thecutaneous lesions clinically and histologically (i.e., with dermalatrophy, re-epithelialization, and stable regression of disease). Fourother patients (3 with LL_(S) and 1 with BL) who did not receivefollow-up treatment with L--Glu--L--Trp showed a worsening of diseasefrom the 6 month to the 1 year observation with new papules and maculesapparent in the skin, and new perivascular granulomas evidenthistologically. The HI and EI values for these patients showeddeterioration from the 6 month values.

On long-term follow up, one of the four ambulatory patients experienceda relapse of disease, but as a borderline tuberculoid form of leprosy(not lepromatous) and at three weeks after termination of theL--Glu--L--Trp trial.

Both parenteral (im) and intranasal (drops) administration appeareffective. In certain patients nasal stuffiness was encountered withvisible nasal edema, and nasal administration was discontinued.

Materials and Methods:

Patient Examination: Examinations were conducted before treatment, andat time intervals of: i) 5-7 days after the L--Glu--L--Trp treatment,ii) 1 month, iii) 3 months, iv) 6 months, and v) 12 months. Methods forskin examination included general observation, morphometry andplanimetry to quantify the size of cutaneous leprotic lesions (as wellas infiltrates, macules, and lepromas). Patient blood samples (obtainedat the latter observation points) were tested (using standard methods)to determine total protein, creatinine, urea, and hepatic function(i.e., bilirubin, thymol, sulem, and AST and ALT transaminases). Whenappropriate, urinalysis was also conducted.

Indices of Cellular Immunity: Functional activity of T-lymphocytes wasdetermined by microscopically assessing blast transformation ofperipheral blood lymphocytes, i.e., prepared by Ficoll-Urotrast (orFicoll-Paque, Pharmacia, Sweden) density gradient sedimentation.Lymphocytes capable of blast transformation with PHA, Con A, orsynthetic M. leprae antigen were counted microscopically after 36-48hrs. in vitro culture. Lymphocyte subpopulations were determined byimmunofluorescence microscopy using OKT-specific monoclonal antibodies(Ortho, Piscataway, N.J.) and FITC-labeled swine and mouse Ig.

Indices of Humoral Immunity: Humoral immunity to M. leprae wasdetermined by testing for the presence of IgG or IgM in the patientserum using the immunofluorescence agglutination (IFA) method. Briefly,patient antibodies capable of cross-linking bacilli can induceagglutination and after washing the bound antibodies can be visualizedby immunofluorescence microscopy using FITC-labeled anti-human IgG orIgM. Antibodies to M. leprae were also assessed using indirecthemagglutination (of bacilli), radial immunodiffusion in agarose (i.e.,Mancini's), and ELISA assays for M. leprae--specific phenol glycolipidantigen (PGL-1).

Histologic Examination: Lepromatous lesions in patient skin weremicroscopically examined in using cryostat sections or paraffin sectionsof formalin-fixed punch biopsy samples (1×0.5×0.5 cm) using Sudan IIIstaining to reveal neutral lipids, hematoxylin and eosin to visualizeimmune cells, and Sil-Nilssen acid fast staining to reveal M. lepraebacilli. Non-specific acid esterase was determined by fixing biopsysamples with calcium formol (per Becker's method) and staining accordingto the Naohlas, Seligman modification of Gomori's method. An ocularmicrometer was used to measure (in serial sections) the area occupied byleporatomous lesions, as well as the size of the cellular infiltrate. Ascale from 1+ to 6+ was used to score the extent of the tissue sectionthat was saturated with mycobacteria, referred to as the bacterial index(BI). Staging of granulomatous lesions was according to Ridley andJopling: i.e., the histological index (HI)=X+BIG, where X is the decimallogarithm of the granulomatous areas measured in 5-6 serial sections,and BIG is the mean bacterial index of the 5-6 serial sections.Non-specific acid esterase was used to identify activated macrophages ingranulomas in tissue sections and the intensity of the reaction in theepidermis was used to score the stage of the leprous infection: i.e., anesterase index value <1.0 corresponded to a highly active poorlydifferentiated leprotic process; >1.0 to a more granulomatous(developed) mass in the tissue section. Electron microscopy wasconducted on ultra-thin sections from seven selected patients before andafter treatment with L--Glu--L--Trp. Biopsy tissues were fixed inglutaraldehyde and osmium, dehydrated in ethanol and propylene oxide,and embedded in epoxide resin. Leprous infection in skin lesions wasassessed using wound exudates. Exudates were collected from the moistsurface of ulcerative defects, smears on slides were fixed with methanoland stained with azure-eosin according to the method ofRomanovsky-Giemsa. Neutrophil activity was histochemically identifiedusing lysosome cation and HCT tests.

EXAMPLE 19 Malaria: Plasmodium falciparum

Summary Overview: Fifty-four patients (54) with relapsing forms oftropical malaria were entered into the trial. The patients' symptomsranged from moderate to severe. Thirty-three patients (33) were enteredinto the L--Glu--L--Trp treatment group and 21 patients into a controlgroup. L--Glu--L--Trp was administered as a single daily im injection ata dose of 100 μg, or alternatively, in at 1 μg/kg body weight, on eachof 5-10 consecutive days. The treatment resulted in a reduction of thepatients' hepatolineal syndrome, a normalization of hematological andimmunological indices, a reduction of fever, and a decreased incidenceof disease relapses in the treated subjects relative to the controls.The clinical investigators report as follows: "A commission consistingof Asst. Chief of the 175th MH," (Medical Hospital; name omitted) "andthe Chief of the Infection Dept. of the 175th MH," (name omitted) "andthe physicians," (two names omitted) "confirms that L--Glu--L--Trp use(sic) in combination with basic therapy for treating patients withtropical malaria in recurring form with a moderate or severe degree ofseverity hinders the development of early (over the course of 28 days)relapses of disease, and makes for a decrease (normalization) of themeasurements of liver and spleen, as well as an elevation (normalizationof the number of leukocytes)." The hematological, immunologic, andclinical chemistry values recorded in these studies are summarized inTABLES 43-44.

                                      TABLE 43                                    __________________________________________________________________________    Hematology Values                                                                           Normal Before Conventional                                                                          L-Glu-L-Trp                                             Healthy                                                                              Therapy                                                                              Therapy Control                                                                       Therapy                                   Index         Values (n = 20)                                                                      (n = 40)                                                                             (n = 21)                                                                              (n = 33)                                  __________________________________________________________________________    Clotting time (whole                                                                        123 ± 2.1                                                                         102 ± 3.1                                                                         135 ± 3.3                                                                          141 ± 2.4                              blood)                                                                        Kaolin clotting time                                                                        53.6 ± 0.72                                                                       49.8 ± 0.8                                                                        57 ± 0.4                                                                           58 ± 0.3                               PTT           22 ± 0.31                                                                         21 ± 0.37                                                                         21.6 ± 0.4                                                                         23 ± 0.2                               Thrombotest   30.7 ± 0.56                                                                       29.8 ± 0.37                                                                       31 ± 0.7                                                                           33 ± 0.5                               Fibrinogen Concentration (g/L)                                                              2.74 ± 0.56                                                                       3.2 ± 0.37                                                                        3.0 ± 0.36                                                                         3.4 ± 0.1                              FDP (mcg/ml)  --     64 ± 2.2                                                                          45 ± 2.14                                                                          23 ± 1.8                               ATIII (%)     100 ± 1.2                                                                         81 ± 1.1                                                                          82 ± 0.4                                                                           98 ± 1.38                              Total Fibrinogen                                                                            154 ± 12.6                                                                        200 ± 7.0                                                                         196 ± 6.6                                                                          200 ± 2.7                              Hagemann factor dependent                                                                   14.4 ± 1.2                                                                        39 ± 3.6                                                                          35.9 ± 2.6                                                                         25 ± 2.58                              fibrinolysis                                                                  __________________________________________________________________________

The results presented in TABLE 43 show that this malaria trialpopulation exhibited no overt signs of a coagulation abnormality (i.e.,as measured by clotting time, PTT, and Thrombotest), but did exhibitelevated levels of fibrinopeptide (FDP) and slightly decreased levels ofantithrombin III (ATIII) suggesting some level of underlying thrombosis.While L--Glu--L--Trp treatment did not alter coagulation values, thelevels of FDP decreased and ATIII increased, suggesting an effect on theunderlying thrombotic processes in these patients. (These findings aresimilar to those presented in Examples 4 and 6, above.)

The levels of the following acute phase reactant proteins were evaluatedin the trial subjects before and after L--Glu--L--Trp, or control,treatment and comparisons were made to the levels in normal healthysubjects: complement C3, prealbumin, ceruloplasmin, orosomucoid, α₂-macroglobulin, and transferrin. The results showed that the patients inthe trial population exhibited no elevation in acute phase reactantsprior to treatment, and after L--Glu--L--Trp (or control) treatments nostatistically significant change in these parameters was observed.

                                      TABLE 44                                    __________________________________________________________________________    Immunology Values:                                                                     Normal Before Conventional                                                                           L-Glu-L-Trp                                            Healthy Values                                                                       Therapy                                                                              Therapy  Therapy                                       Index    (n = 20)                                                                             (n = 40)                                                                             Control (n = 21)                                                                       (n = 33)                                      __________________________________________________________________________    Leukocytes:                                                                   (× 10.sup.9 /L):                                                                 5.8 ± 0.25                                                                        6.6 ± 0.1                                                                         3.7 ± 0.1                                                                           9.0 ± 0.14                                 % Normal Value:                                                                        (100%) (114%) (64%)    (155%)                                        Lymphocytes:                                                                  (× 10.sup.9 /L):                                                                 1.74 ± 0.1                                                                        1.90 ± 0.07                                                                       1.68 ± 0.03                                                                         2.57 ± 0.09                                % Normal Value:                                                                        (100%) (109%) (97%)    (148%)                                        T-Lymphocytes:                                                                (× 10.sup.9 /L):                                                                 0.89 ± 0.1                                                                        0.55 ± 0.04                                                                       0.50 ± 0.01                                                                         0.94 ± 0.06                                % Normal Value:                                                                        (100%) (62%)  (57%)    (106%)                                        B-Lymphocytes:                                                                (× 10.sup.9 /L):                                                                 0.46 ± 0.02                                                                       0.36 ± 0.01                                                                       0.32 ± 0.02                                                                         0.53 ± 0.02                                % Normal Value:                                                                        (100%) (77%)  (70%)    (115%)                                        IgG (mg/ml):                                                                           134 ± 5.6                                                                         143 ± 7.3                                                                         146 ± 4.4                                                                           187 ± 1.9                                  % Normal Value:                                                                        (100%) (94%)  (109%)   (140%)                                        IgA (mg/ml):                                                                           98.3 ± 6.2                                                                        140 ± 5.1                                                                         159 ± 5.11                                                                          138 ± 6.0                                  % Normal Value:                                                                        (100%) (143%) (162%)   (140%)                                        IgM (mg/ml):                                                                           127 ± 9.7                                                                         153 ± 5.9                                                                         179 ± 4.0                                                                           193 ± 9.0                                  % Normal Value:                                                                        (100%) (120%) (141%)   (152%)                                        __________________________________________________________________________

The results presented in TABLE 44, show a slight elevation in totalperipheral blood leukocytes, and T- and B-lymphocytes in this trialgroup prior to therapy, with still further decreased PBL counts (and T-and B-lymphocyte counts) being observed following conventional treatmentwith anti-malarial agents (e.g., quinine derivatives) that have bonemarrow suppressive effects. Treatment with L--Glu--L--Trp increased PBLand T- and B-lymphocyte counts to normal or supra-normal values. Theincreases in B-lymnphocyte counts in L--Glu--L--Trp treated patientswere accompanied by increased serum levels of IgG and IgM.

EXAMPLE 20 Hemorrhagic Dengue Fever

Protocol A

Summary Overview: L--Glu--L--Trp was administered to 21 patients withhemorrhagic Dengue Fever, and 28 patients receiving conventional therapyserved as controls. L--Glu--L--Trp was administered im single dailydoses of 100 μg on each of 5 consecutive days as an adjunct to ongoingconventional therapy. Treatment with L--Glu--L--Trp resulted in decreasein fever, reduction of toxic symptoms (i.e., chills, headache, etc.),significant decrease in hepato-lineal syndrome. The investigators alsoobserved that L--Glu--L--Trp treated patients experienced a reduction inmuscle and bone pain. Immunological indices in peripheral blood werealso normalized.

Patient Population: Forty nine patients (20-30 years of age; Kinhnationality) with diagnosed Dengue Fever were entered into the trial.The patients were referred to the 175th Military Hospital in Ho Chi MinhCity at the time of an epidemic of Dengue fever in the (then) SouthSoviet Republic of Vietnam. Diagnosis was based on the criteriarecommended by the World Health Organization (1986) using medicalhistories, and clinical and laboratory data. Serological analyses wereconducted at the Arbovirus Infectious Diseases Laboratory of the PasteurInstitute in Ho Chi Minh City. All patients had a first or second degreedisease severity. Clinical indicia of disease activity included: fever,chills, headache (98.3% of patients), dizziness (95%), weakness (100%),loss of appetite (95%), sleep disturbance (90%), sensations of mouth andlip dryness (85%), constipation (48.3%), catarrh in the nasopharynx withcough (35%), hyperemic facial tissue with pain in the superior ocularorbit (58.6%), inflammation in scleral vessels (55.2%), lightsensitivity (32.2%), and muffled heart tones (86.7%) or a functionalsystolic murmur auscultated on the apex cordis (5.7%). Dyspeptictoxicosis, i.e., nausea and vomiting were rarely encountered (15% and11.7%, respectively). Joint and muscle pains were pronounced, mostcommonly involving the spinal joints in the lumbar region (96.6%),joints of the pelvic girdle (82.2%), the ulnar joints (53.4%), as wellas, the muscles of the lumbar (69%) and abdominal regions (69%).Exanthema (40.7%) and lymphadenopathy (46.7%) were evident in certainpatients. Dermal puritus with rash was seldom observed (6.1%), buthepatomegaly (65.5%) and splenomegaly (53.4%) were pronounced, as wereleukopenia, thrombocytopenia, and changes in blood coagulation time(during periods of hemorrhaging induced by the virus). Leukocytes duringpeak episodes of disease were recorded to be 4.60±0.17×10⁹ /L, and theabsolute number of lymphocytes varied widely from 0.95 to 4.29×10⁹ /L.

Patients were divided into two groups: Group 1; consisting of 28patients ("Control") treated only with conventional maintenance therapy;and, Group 2, consisting of 21 patients treated with L--Glu--L--Trp incombination with conventional maintenance therapy.

Treatment Regimens: Group 1 received conventional maintenance therapyconsisting of detoxifying preparations and methods, e.g., fluids, fevercontrol agents and the like. Group 2 received combination therapy, i.e.,maintenance therapy and L--Glu--L--Trp, but only during periods of acutedisease activity. L--Glu--L--Trp was administered at a dosage of 500 μg.The efficacy of the respective treatment protocols was evaluated byrecording the most frequently used clinical indicia of Dengue diseaseactivity: namely, the clinical indicia recorded in TABLE 45.

                  TABLE 45                                                        ______________________________________                                        Primary Symptoms of Hemorrhagic Dengue Fever in Patients Treated              with Conventional Therapy in Combination with L-Glu-L-Trp- Protocol A                     Duration of Symptom(s) in Hours:                                                Conventional                                                                             Combination                                                        Therapy    L-Glu-L-Trp Therapy                                  Clinical Index                                                                              (n = 28)   (n = 21)                                             ______________________________________                                        Fever:                                                                        At day 2-6 of illness:                                                                      7.3 ± 0.6                                                                             5.2 ± 0.3††                         At day 2-4 of illness:                                                                      7.3 ± 0.6                                                                             4.7 ± 0.2†††                 Headache:     12.2 ± 1.1                                                                            8.1 ± 0.9†                                 Weakness:     11.6 ± 1.2                                                                            9.3 ± 0.8†                                 Loss of appetite:                                                                           11.4 ± 1.1                                                                            7.5 ± 0.8††                         Insomnia:     11.0 ± 1.3                                                                            7.1 ± 0.7†                                 Hepatomegally:                                                                              15.8 ± 0.6                                                                            11.3 ± 0.8†††                Splenomegally:                                                                              13.3 ± 0.7                                                                            9.6 ± 0.7††                         Lumbar pain:  13.8 ± 1.4                                                                            8.7 ± 0.7††                         Joint pain:   12.2 ± 1.5                                                                            7.4 ± 0.7††                         Waist/Pelvic girdle pain:                                                                   12.7 ± 1.3                                                                            8.1 ± 0.8†                                 Skeletal muscle pain:                                                                       11.2 ± 1.7                                                                            7.7 ± 1.1                                         Eye muscle pain:                                                                            11.0 ± 1.1                                                                            6.6 ± 0.8††                         ______________________________________                                         †, p < 0.05; ††, p < 0.01; †††,     p < 0.001                                                                

Summary of Clinical Disease Responses in the Patients--Protocol A:Therapeutic efficacy of L--Glu--L--Trp was subjectively observed inthese studies even before the collation of the objective evidence(above). In most patients, the day after initiating L--Glu--L--Trptherapy, body temperature became normal, and those patients who did notnormalize on the first day normalized on day 2. A direct correlation wasnoted between the length of fever and the disease free intervalfollowing fever. L--Glu--L--Trp treatment appeared to increase theabsolute numbers of leukocytes and T-lymphocytes. No side effects orallergic effects were observed during L--Glu--L--Trp treatment.L--Glu--L--Trp treatment led to a more rapid convalescence andrestoration of the subjects' ability to perform work.

Protocol B

Summary Overview: Sixty Dengue virus infected patients (60) were enteredinto the trial; 36 were treated with L--Glu--L--Trp and 24 were treatedusing conventional therapy (i.e., controls). Administration ofL--Glu--L--Trp at a dosage of 100 μg im, or alternatively 1 μg/kg in, oneach of 5-10 consecutive days resulted in the reduction of fever, morerapid reduction of toxic symptoms (e.g., chills, fever, rash, headaches,muscle aches and pains) and the restoration of immunologic indices.

EXAMPLE 21 Infectious Disease: Tuberculosis

Early Results: Sixty-three patients having pulmonary infection withMycobacterium tuberculosis (TB) were entered into the trial: 37 patientswere treated with L--Glu--L--Trp and 26 patients (controls) were treatedusing conventional treatments. L--Glu--L--Trp was administered fivetimes at a dosage of 50 μg to 100 μg every other day and as an adjunctto treatments with conventional antibiotics. Clinical evaluation of thepatients at 2 months post-treatment with L--Glu--L--Trp revealed adisappearance of toxic symptoms, a resorption of pulmonary infiltrates,and a resolution of pulmonary cavities. The disappearance of TB bacilliwas noted in the sputum. Prior to treatment, immune parameters inperipheral blood were abnormal, i.e., decreased, and following treatmentwith L--Glu--L--Trp these parameters were normalized.

Expanded Results: One hundred and five patients (105) were entered intothe trial. The patients, aged 19 to 60 years, consisted of 73 men and 32women who were presenting for the first time with active progressivepulmonary TB infection. All patients in the trial had been hospitalizedbecause of the severity of their disease. According to standarddiagnostic criteria at clinical presentation: 59 patients had signs ofpulmonary infiltration; 11 showed evidence of disseminated lung disease;10 exhibited at least one fibrous/cavernous foci of infection; 9 showedat least one cavernous foci; and 16 had tuberculomas. The 105 patientswere divided into three groups as follows: namely, Group 1, 37 patients,treated with conventional therapy and L--Glu--L--Trp (i.e., 50-100 μgdaily for 5 days); Group 2, 22 patients, treated with conventionaltherapy and the immunomodulator "decaris" (i.e., 150 μg, twice weekly,for 1.5-2 months); and, Group 3, consisted of 46 patients, who receivedonly conventional therapy (i.e., no immunomodulators).

Clinical criteria used in evaluating patients' responses included i)"considerable improvement," ii) "improvement," and iii) "no change"which were objectively defined from patient examinations and evaluationof chest X-ray data as follows: "Considerable improvement" requiredthat 1) wheezing and catarrhal phenomena disappear completely from thelungs; 2) mononuclear cell infiltration and degenerative foci in thelungs largely disappear; 3) cavities of tissue degeneration in the lungdisappear; nd 4) expectoration of acid fast bacteria cease;"Improvement" required that 1) wheezing (only) disappear from the lungs;2) a progressive resolution of cellular infiltration and degenerativefoci in the lungs; and, 3) a partial consolidation of degenerative foci;and 4) a reduction in the size of cavities in the lung.

The results of clinical evaluation at 2 months of therapy are summarizedin TABLE 46.

                  TABLE 46                                                        ______________________________________                                        Effects of Treatment with L-Glu-L-Trp on Clinical Parameters                  of Lung Disease in Patients of with Progressive Tuberculosis: 2 months              No.    Conventional                                                                              No     Im-    Considerable                                 Pa-    Treatment* in                                                                             Change provement                                                                            Improvement                            Group tients Combination with                                                                          No.  %   No.  %   No.  %                             ______________________________________                                        1     37     L-Glu-L-Trp 1     3  27   73  9    24                            2     22     decans      3    13  18   82  1    5                             3     46     none        7    15  36   78  3    6                             ______________________________________                                         *C, conventional antibiotic therapy; d, "decaris" immunomodulator.       

The results presented in TABLE 46 show the following: in Group 1(combination therapy with L--Glu--L--Trp), "considerable improvement"was observed in 9 patients (24%) and an additional 27 patients showed"improvement," i.e., for a total response rate of 97% of the patientsshowing improvement. (Degeneration foci and cavities in the lung wereclosed in the 9 patients responding rapidly to therapy.); in Group 2("decaris" immunomodulator combined therapy), only one patient(5%)showed "considerable improvement" and "improvement" was observed in18 patients, i.e., for a response rate of 86%; and, in Group 3(conventional therapy only), "considerable improvement" was noted in 3patients (6%) and "improvement" in 36, i.e., for a response rate of 84%.

At 4-6 months of in-hospital treatment: in Group 1, positive changeswere noted in the lungs of 35 of the 37 patients, i.e., closure ofdegeneration foci and cavities. One patient was discharged from thetrial for a procedural violation, and one patient exhibited diseaseprogression of what was determined later to be an antibiotic resistantstrain of M. tuberculosis; in Group 2, foci and cavities of degenerationin the lungs were closed in 10 patients of the 22 in the trial (45%),and in Group 3, foci and cavities of degeneration in the lungs wereclosed in 14 patients of the 46 in the trial (48%). Patients toleratedL--Glu--L--Trp therapy well, and no toxicity or allergic reactions wereobserved

EXAMPLE 22 Respiratory Disease: Asthma and Allergy

Protocol A

Summary Overview: Sixty five patients (65) with bronchial asthma,including both adults and children, were entered into the trial: 37patients were treated with L--Glu--L--Trp and 28 (controls) receivedconventional therapy alone. L--Glu--L--Trp was administered im in asingle daily dose of 1 μg/kg on each of 5-10 consecutive days. Treatmentwith L--Glu--L--Trp resulted in less severe asthmatic clinical symptoms,a decrease in the requisite duration of medical treatment, and asignificant reduction in the incidence of bronchial obstruction andlaryngotracheitis relative to the control group. In certain patients itwas possible to eliminate the use of prescribed steroids. In the yearlong follow-up period of clinical observation, a 4.2-fold decrease inthe incidence of bronchial asthmatic attacks was observed in theL--Glu--L--Trp treated subjects. A disappearance of clinical symptoms offood and drug allergies was observed in more than half of theL--Glu--L--Trp treated patients.

Protocol B

The use of L--Glu--L--Trp as an ingredient or applicant in cosmeticformulations and preparations provides less allergenic materials and/ormaterials evoking a lesser degree of allergic reactions in sensitizedsubjects.

Protocol C

Patient Population: Twenty-eight children (19 boys; 9 girls) wereentered into the trial for treatment with L--Glu--L--Trp: 17 were aged1-3 years; 8 were aged 4-7 years; and, 3 were aged >8 years. Patientspresented with the following diagnoses: 14 with acute respiratory viralinfections (ARVI) and bronchitis; 4 with acute respiratory infection(ARI) complicated with recurrent purulent otitis; 2 with ARI andprolonged sub-febrile infection; and, 11 with ARI and lymph nodeenlargement and hyperplasia of the palatine and nasopharyngeal tonsils.Bronchial asthma was diagnosed in 6 children and obstructive bronchitisin 8. In 11 children there was evidence, or medical history of,exudative/catarrhal diathesis; in 4 there was lymphatic/hypoplasticdiathesis. The patient population was also selected for medical historyof allergy, and 6 patients had one or more medicinal allergies, 7 hadfood allergies, 5 had both a food and a medicinal allergy, and one childhad a history of bronchospasms initiated by chrysanthemums. Of thechildren 15 had one or more family members with a history of allergy,asthma, hayfever, or medicinal allergy. All patients had a previousmedical history of recurrent infections and had previously receivedrepeated courses of antibacterial preparations, i.e., penicillin-basedantibiotics, sulfanilamides (biseptol), gammaglobulins, andphytotherapy.

A comparison (control) group consisted of 73 frequently ill childrenaged 1-7 years and including 44 boys and 29 girls.

Treatment Protocol: The dosage of L--Glu--L--Trp administered to thesechildren was as follows: children 1-3 years of age received 20 μg; 4-5years of age received 30 μg; 6-7 years of age received 40 μg; and,those >7 years of age received 50 μg. The L--Glu--L--Trp preparation wasadministered im daily for 5 days and a repeat course of therapy wasadministered at 34 months. Two treatment courses were required forsuccessful therapy. (Two children with pronounced allergies receivedZaditen, and one of the two also received histoglobulin.)

Indicators Evaluated: The patients' response to therapy was evaluated bymonitoring: physical condition (i.e., by physical examination), bodytemperature, blood chemistry, proteins, hematology, and immunology.

Immunological examination showed lower T-lymphocyte cell counts inpatients prior to treatment than in normal healthy children, with adecrease noted in "active" T-lymphocytes, i.e., as measured by adecrease in the number of theophylline resistant lymphocytes ("active")without a change in the number of theophylline sensitive lymphocytes("quiescent").

In vitro incubation of lymphocytes, (prepared from thirty of thepatients prior to treatment) with L--Glu--L--Trp showed an increase inthe percentage of E-rosette forming (CD2⁺) T-lymphocytes in therespective different patient lymphocyte populations. Parallel incubationwith the immunomodulator T-activin did not effect a change in thepercentage of positive cells.

Following the first course of L--Glu--L--Trp therapy a statisticallysignificant increase was noted in both the absolute number andpercentage of T-lymphocytes in peripheral blood. A increase in thenumber of theophylline-resistant lymphocytes was also observed, with aslight decrease in the number of theophylline-sensitive lymphocytes.After the second course of L--Glu--L--Trp immunologic parameters were inthe normal range.

Clinical evaluation of patients over 6-12 months documented a decreasein the number of respiratory infections in 92.9% of the children treatedwith L--Glu--L--Trp, with an average reduction in the number ofinfections 3.6-fold, and reduction in attacks of bronchial asthma (inthe asthma patients) by 4.2-fold. Courses of illness were also milder(than pre-treatment illnesses) and did not require hospitalization.Infections in the treated patients were not accompanied by complicationwith bronchial obstruction or otitis. The average length of illness wasdecreased by 4-5 days, and food allergies disappeared in 67.8% of thepatients having this pre-existing condition at the beginning of therapy.Patients experienced an increase in physical activity and appetite, withdecrease in subfebrile episodes and manifestations of lymphadenopathy.

EXAMPLE 23 Gastro-Intestinal Disease

Protocol A Gastric Ulcers

Gastric ulceration is accompanied by a persistent immunodeficiencycondition in the pre-ulcerative stage, i.e., as the ulcer is developingin patients with gastritis.

Patient Population: Twenty two patients (all men aged 18-30 years)suffering from chronic primary gastritis and gastro-duodenitis wereentered into the trial. Diagnosis was based on physical examination, andclinical and laboratory investigations including roentgenoscopy andendoscopic examination of the stomach and duodenum. Patient entrycriteria was further conditioned on the results of laboratory tests fori) pretreatment immune indices (i.e., numbers of lymphocytes, T-, andB-lymphocytes, etc.; TABLE 46) and ii) the in vitro sheep red blood cellrosette forming response of T-lymphocytes to L--Glu--L--Trp, (i.e.,whether in vitro incubation of patient peripheral blood lymphocytes withL--Glu--L--Trp increased the percentage of E-RFC). Ten patients (45%)exhibited a negative in vitro response to L--Glu--L--Trp and alsoexhibited immune indices that were within the normal range, and thesepatients were excluded from the trial. Twelve patients (55%) exhibited apositive in vitro response to L--Glu--L--Trp and immune indices thatwere altered, and outside the normal range, i.e., low absoluteT-lymphocyte counts and increased absolute numbers of Null-lymphocytes.These twelve patients were entered into the trial.

For determining normal values, the control group consisted of 28 normalhealthy volunteers aged 17-23.

Treatment Protocol: Patients received L--Glu--L--Trp im daily at adosage of 100 μg for 5 days in combination with their conventionalongoing treatment.

Laboratory Tests: Lymphocyte sub-populations before and after treatmentare presented in TABLE 47, in comparison with the results recorded withnormal healthy subjects.

                                      TABLE 47                                    __________________________________________________________________________    Immunology Values                                                                       Normal Healthy                                                                        Before After L-Glu-L-Trp Combination                        Index     Values  Therapy                                                                              Therapy                                              __________________________________________________________________________    T-Lymphocytes (%):                                                                      59.3 ± 2.4                                                                         44.0± 2.2††                                                         55.4 ± 2.1**                                      (× 10.sup.9 /L):                                                                  1.087 ± 0.07                                                                       0.869 ± 0.44†                                                              1.04 ± 0.04*                                      % Normal Value:                                                                         (100%)  (80%)  (96%)                                                B-Lymphocytes (%):                                                                      8.0 ± 1.0                                                                          7.5 ± 0.8                                                                         7.2 ± 1.5                                         (× 10.sup.9 /L):                                                                  0.145 ± 0.02                                                                       0.153 ± 0.02                                                                      0.14 ± 0.02                                       % Normal Value:                                                                         (100%)  (106%) (97%)                                                Null Lymphocytes (%)                                                                    31.5 ± 2.2                                                                         48.5 ± 1.9††                                                        37.3 ± 2.0**                                      (× 10.sup.9 /L):                                                                  0.621 ± 0.07                                                                       0.95 ± 0.09†                                                               0.9 ± 0.05*                                       % Normal Value:                                                                         (100%)  (153%) (145%)                                               __________________________________________________________________________     †, p < 0.05 in comparison with normal; ††, p < 0.001     in comparison with normal; *, p < 0.01 in comparison with pretreatment        values; **, p < 0.001 in comparison with pretreatment values.            

The results presented in TABLE 47 show that treatment withL--Glu--L--Trp induced a statistically significant increase in thepercentage and absolute numbers of T-lymphocytes, with a concomitantfall in the percentage and numbers of Null-lymphocytes.

Clinical Response: Treatment of the patients in the trial populationwith L--Glu--L--Trp lead to a more rapid recovery and the patientsachieved a stable remission of chronic gastritis and gastroenteritis.

Protocol B Infectious Disease: Shigella dysenterae

Summary Overview: A total 125 patients infected with Shigella dysenterywere examined. Fifty-three patients constituted the control group.L--Glu--L--Trp was administered im single doses of 100 ug for 10consecutive days with resultant normalization of fever, reduction oftoxemia, and normalization of gastrointestinal disorders and symptoms.Bacterial shedding in the GI track was observed to cease, and theimmunological indices were normalized.

EXAMPLE 24 Acquired Immunodeficiency: Adult Thymectomy

Summary Overview: A total of 12 thymectomized patients were treated withL--Glu--L--Trp. Prior to therapy, these individuals had experiencedfrequent serious infections including upper respiratory infections.L--Glu--L--Trp was administered in a single dose of 100 μg daily for 10days and repeated every 4-6 months. The normalization of immunologicindices was observed, and there was a reduction of infectious disordersincluding cutaneous infections and other chronic exacerbations.

EXAMPLE 25 Hepatic Diseases

Protocol A Jaundice and Systemic Toxicity

Summary Overview: Thirty four patients (34) with diagnosed chronic liverdisease were treated with L--Glu--L--Trp (Group 1). Entry into the trialwas established on the basis of clinical symptoms and serumbiochemistry, i.e., levels of aminotransferase, bilirubin,gamma-globulins, and thymol test results, as well as the results ofradioisotope and ultrasound scanning. In certain cases liver biopsyspecimens were obtained, and confirmed the diagnosis. Patient entry intothe trial was also occasioned by symptoms and either i) presence of anabnormal T-helper/T-suppressor ratio, or ii) detectable levels ofAustralia antigen in serum. Twenty seven patients (27) fulfilling thesame diagnostic criteria were entered into a control group forconventional treatments (Group 2).

Immune status was assessed by collecting and testing samples ofperipheral blood. The following immune parameters were tested: totalnumber of T-lymphocytes (E-RFC), theophylline-resistant lymphocytes(T-helpers), and theophyline-sensitive lymphocytes (T-suppressors);activity of Fc-receptor lymphocytes in antibody-dependent cellularcytotoxicity assays; serum concentrations of IgA, IgM, IgG, and IgD;and, the level of circulating immune complexes.

L--Glu--L--Trp was administered daily im at a dose of 100 μg on each of5 to 10 consecutive days.

At time of entry into the trial, patients in Group 1 presented with thefollowing: 29 patients were seropositive for hepatitis virus (i.e., 26patients had circulating Hepatitis B antigen and 3 had circulatingautoantibodies); 5 patients had cirrhotic liver disease (3 of viraletiology and 2 or unknown origin) with micronodular and mixed initialstage forms of disease. (L--Glu--L--Trp was not prescribed to patientswith dystrophic cirrhotic liver disease.) All patients presented withabnormally decreased levels of T-suppressor cells in peripheral blood.

Clinical Effects: Treatment of the patients in Group 1 withL--Glu--L--Trp resulted in three different types of clinical responses:i) in 21 of the 29 patients with serological evidence of hepatitis virusinfection (above) a positive response to therapy was observed; ii) inthe remaining 8 patients (of the 29 seropositive patients) no change wasdetectable either in the clinical picture or in the serum biochemicalmarkers; and, iii) in 4 patients, (2 having autoantibodies; and 2 withcryptogenic cirrhosis) illness was aggravated.

The 21 patients of Group 1 having a positive response to L--Glu--L--Trptreatments exhibited the following at the conclusion of the 5 days oftherapy: i) decreased symptoms of cellular insufficiency, weakness, andheadache; ii) improved serum biochemical markers (i.e., bilirubin andaminotransferase activity); iii) improved ability to sleep; and, iv)decreased pain in the hypochondrium. In 2 of the responsive patients,jaundice disappeared with noticeable lessening of telangiectasia in theface. Following treatment, responsive patients showed i) an increase inthe number of peripheral blood T-lymphocytes, (primarily because of anincrease in the number of suppressor T-cells); and, ii) a decrease inthe numbers of undifferentiated Null lymphocytes. In the 3 patients withmixed cirrhotic liver disease of viral etiology, ALT levels in serumdropped, suggesting a positive effect on liver cytology.

The 8 patients in Group 1 having a negative response to L--Glu--L--Trptreatments (i.e., patients with less severe immunodeficiencies andlesser immunoregulatory impairments) exhibited the following at theconclusion of the 5 days of therapy: i) no significant changes inclinical symptoms; ii) no (or minimal) changes in serum biochemicalmarkers, i.e., ALT and bilirubin levels dropped slightly but AST andthymol test results increased slightly); and ii) lymphocyte countsincreased with increases in the number of Null and T-lymphocytes.

The 4 patients of Group 1 having aggravation of disease activityfollowing the L--Glu--L--Trp treatments exhibited the following at theconclusion of the days of therapy: i) increased serum transaminaselevels (AST; ALT); ii) increased thymol assay values; iii) increasedautoantibody levels; iv) increased levels of immunoglobulins; and, v)increased numbers of T-helper lymphocytes.

Patients in the control Group 2 were, as previously, relativelynon-responsive to conventional therapy and showed few signs of clinicalresponse to therapy or changes in serum biochemical markers.

Protocol B Hepatitis

Chronic hepatitis is an urgent problem in pediatric medicine with 350million people worldwide registered to be carriers of the virus, and 2million new cases each year. In 1 out of 10 patients, the viralinfection takes a chronic course with cirrhosis of the liver andultimately a fatal outcome. In 80% of cases of primary hepatoma, a linkwith the hepatitis virus has been recorded.

Patient Population: In accord with a U.S.S.R. Ministry of Healthdecision 125 children suffering with chronic persistent hepatitis B wereentered into this trial over a two month period. Group 1 consisted of 35children who received decaris immunomodulatory therapy; Group 2, 38children who received Thymalin treatments; Group 3, 19 children whoreceived L--Glu--L--Trp; and, Group 4, 32 children who received onlyconventional symptom specific therapy. All patients admitted to thetrial exhibited signs of secondary immunosuppression, as evidenced by adecrease in the numbers of E-rosette forming T-lymphocytes.

Treatment Protocol: L--Glu--L--Trp was administered at a dose of 2 μg/kgof body mass, im, daily for 3 days, and a repeat (identical) course oftreatment was administered 10 days after termination of the firstcourse.

Observations: In about 63% patients at the conclusion of L--Glu--L--Trptreatment, numbers of E-RFC T-lymphocytes were increased and the ADCCactivity (measured in vitro with antibody-coated target cells) wasdecreased to normal levels. A retrospective analysis of the datarevealed two classes of patients:. namely, the 63% responders and theremaining non-responders, i.e., based on differences in clinicalresponses to therapy, serum levels of ALST, E-RFC, ADCC activity, andT4⁺ /T8⁺ ratios. A correlation was noted between the percentage ofnon-responders (than responders) with detectable auto-lymphocytotoxic(ALCT) antibodies (a marker for autoimmune disease in the patients), theindex of ALCT cytotoxic activity, and the failure of L--Glu--L--Trptreatment to influence these markers in non-responder subjects.L--Glu--L--Trp treatment apparently affected the latter markers in thesubgroup of responders.

                                      TABLE 48                                    __________________________________________________________________________    Immunology Values:                                                                      Normal Non-Responders                                                                             Responders                                                Healthy                                                                              Before                                                                              After  Before                                                                              L-Glu-L-Trp                                         Values Therapy                                                                             L-Glu-L-Trp                                                                          Therapy                                                                             Therapy                                   Index     (n = 20)                                                                             (n = 7)                                                                             (n = 7)                                                                              (n = 12)                                                                            (n = 12)                                  __________________________________________________________________________    T-Lymphocytes                                                                 E-RFC) % of PBL:                                                                        61.0 ± 2.70                                                                       42.17 ± 2.4                                                                      56.7 ± 3.24                                                                       34.57 ± 2.57                                                                     47.33 ± 2.2                            (× 10.sup.9 /L):                                                                  1624 ± 58                                                                         770 ± 71                                                                         1382 ± 76                                                                         779 ± 41                                                                         1227 ± 65                              % Normal Value:                                                                         (100%) (47%) (85%)  (48%) (76%)                                     Ratio OKT4.sup.+ /OKT8.sup.+ :                                                          1.33 ± 0.05                                                                       0.82 ± 0.07                                                                      0.82 ± 0.007                                                                      0.90 ± 0.007                                                                     1.08 ± 0.07                            ALCT: % of subjects:                                                                    0      85.4 ± 13.2                                                                      57.14 ± 18.7                                                                      41.67 ± 14                                                                       16.7 ± 13.2                            Cytotoxic Index:                                                                        0      50.4 ± 5.5                                                                       44.55 ± 6.8                                                                       29.9 ± 1.9                                                                       50.4 ± 5.5                             __________________________________________________________________________

Clinical Responses: In patient's lacking an autoimmune disease component(i.e., as measured by ALCT), their disease resolved, while the diseaseof those having an autoimmune component did not. Disease activity wastracked over the six months following therapy, and remission was foundto negatively correlate with the presence of a pre-treatment autoimmunecomponent (i.e., ALCT). One hundred percent of the patients lackingdemonstrable ALCT in the pre-treatment interval exhibited stableremission over the 6 months of observation, as contrasted with rates of80% stable remission in patients with moderate ALCT activity, and 0%stable remission in patients with the highest ALCT levels.

                  TABLE 49                                                        ______________________________________                                                         Percent of Subjects Disease                                        Pre-Treatment                                                                            Free at 6 Months After Treatment With:                       Group ALCT       Conven-                L-Glu-                                #     Activity   tional   Descaris                                                                             Thymalin                                                                             L-Trp                                 ______________________________________                                        1     None       75       100    100    100                                   2     Moderate   35       67     76     80                                    3     High       0        67     33     0                                     ______________________________________                                    

EXAMPLE 26 Effects of L--Glu--L--Trp Dipeptide on Expression ofE-receptors on Thymocytes

L--Glu--L--Trp has been reported to restore T-cell receptors and E-RFCfollowing incubation at 37° C. Thymus cells were isolated from maleguinea pigs (180-200 g) under standard conditions of tissue mincing anddifferential centrifugation in Medium 199. Isolated thymocytes weretreated with trypsin to remove cell surface E-receptors capable ofbinding rabbit erythrocytes (E). The percentage of cells capable formingE-rosettes (E-RFC) was decreased by about two-fold under theseconditions.

Thymocytes were prepared from 48 guinea pigs, treated with trypsin, andtested for E-RFC according to Nekam K., et al., Immunopharmacol5(1):85-94 (1982), incorporated herein by reference.

Dipeptide L--Glu--L--Trp was dissolved in 0.9% (w/v) NaCl and added tocultures at test concentrations of 1 mg/mL, 0.01 mg/mL, and 0.0001mg/mL. The results of these experiments are summarized in TABLE 50.

                  TABLE 50                                                        ______________________________________                                        Effects of L-Glu-L-Trp on Expression of                                       E-receptors on Thyrmocytes (X ± S.D.)                                      Trypsin              Conc.       E-RFC                                        Treatment  Addition  (mg/ml)     (%)                                          ______________________________________                                        -          None      0           66.0 ± 5.4                                +          None      0           26.0 ± 1.8*                               +          L-Glu-L-Trp                                                                             1           42.8 ± 3.5*†                       +          L-Glu-L-Trp                                                                             0.01        36.8 ± 2.4*†                       +          L-Glu-L-Trp                                                                             0.0001      30.3 ± 3.1*                               ______________________________________                                         *, indicates significance at the p < 0.05 level in comparison with the        nontrypsin treated control values ("-"/None/0); and                           †, indicates significance at the p < 0.05 level in comparison with     the trypin treated control values ("+"/None/0).                          

The results presented in TABLE 50 show that addition of L--Glu--L--Trpat 0.01-1mg/mL increased the percentage of E-RFC in trypsin-treatedthymocyte cultures. The results suggest that L--Glu--L--Trp stimulatesexpression of E-receptors on thymocytes over a wide range ofconcentrations, perhaps by direct ligand-receptor interaction ofL--Glu--L--Trp with the E-rosette receptor.

EXAMPLE 27 Effects on Thymocytes from Aged Animals

Effects of L--Glu--L--Trp on expression of E-receptors on "aged"thymocytes was investigated in a manner similar to that in EXAMPLE 26,above, but using thymocytes obtained from old male guinea pigs (700-800g). In aged animals it is recognized that the percentage of thymocytesforming rosettes with rabbit erythrocytes is reduced, and cell surfaceE-receptors are likewise reduced. For these studies aged thymocytes wereisolated in Medium 199 from 42 guinea pigs, and E-RFC determined asdescribed by Stadecker, M. J., et al., J. Immunol 111(8):4061-4065(1973), incorporated herein by reference. L--Glu--L--Trp was dissolvedin 0.9% (w/v) NaCl and added to cultures of thymocytes at concentrationsof 1, 0.01, and 0.0001 mg/ml. The results of these experiments aresummarized in TABLE 51.

                  TABLE 51                                                        ______________________________________                                        Effects of L-Glu-L-Trp on Expression of                                       E-receptors on Aged Thymocytes (X ± S.D.)                                                Conc.     E-RFC                                                 Addition      (mg/ml)   (%)                                                   ______________________________________                                        None          0         45.3 ± 3.7                                         L-Glu-L-Trp   1          54.6 ± 4.2*                                       L-Glu-L-Trp   0.01      47.8 ± 3.9                                         L-Glu-L-Trp   0.0001    50.3 ± 4.2                                         ______________________________________                                         *, indicates significance at the p < 0.05 level in comparison with the        nontreated control values (None/0).                                      

The results presented in TABLE 51 show, as expected, a reduction in thepercentage of E-RFC in populations of aged thymocytes, i.e., from 66±5%(TABLE 62) to 45.3±3.7% (TABLE 51). L--Glu--L--Trp fostered astatistically significant increase in the percentage of E-RFC at 1mg/mL.

EXAMPLE 28 Effects of Dipeptide L--Glu--L--Trp on T-Lymphocyte Subsetsin Human Peripheral Blood

The effects of L--Glu--L--Trp on human peripheral blood T-lymphocyteswas investigated using indirect immunofluorescence microscopy and OKT4and OKT8 monoclonal antibodies (Ortho, USA) directed to lymphocyte cellsurface differentiation antigens. For these studies lymphocytes wereisolated from heparinized peripheral blood (25 units heparin/mL)isolated from 18 different donors with inflammatory lung diseases andchronic purulent bronchitis accompanied by a secondary immunodeficiencystate. Lymphocytes were prepared by centrifugation on Ficoll-Hypaque,washed, and then incubated in vitro for 45 minutes at 37° C. in thepresence (or absence) of L--Glu--L--Trp at concentrations of 1 mg/mL,0.01 mg/mL, 0.0001 mg/mL. Following incubation the cells were washedwith Medium 199 and the percentage of OKT4⁺ (T-helper) and OKT8⁺(T-suppressor) lymphocytes determined by indirect immunofluorescentmicroscopy. The results presented in TABLE 51 show the mean percentagesof OKT4⁺ and OKT8⁺ cells in this group of patients after incubation withMedium 199 (Control), or with L--Glu--L--Trp. The OKT4⁺ /OKT8⁺ ratiosfor this group of patients is also shown in TABLE 52.

                  TABLE 52                                                        ______________________________________                                        Effects of L-Glu-L-Trp on T-helper (OKT4.sup.+)                               and T-suppressor (OKT8.sup.+) Lymphocytes from Patients                       with Secondary Immunodeficiency (X ± S.D.)                                 Dose                                                                          (mg/ml)       Control  L-Glu-L-Trp                                            ______________________________________                                        (OKT4.sup.+)                                                                  1             23.9 ± 2.1                                                                           39.7 ± 2.7*                                        0.01          2.42 ± 1.7                                                                           39.8 ± 2.4*                                        0.0001        25.1 ± 0.1                                                                          25.4 ± 2.3                                          (OKT8.sup.+)                                                                  1             19.9 ± 1.9                                                                          21.4 ± 1.3                                          0.01          20.6 ± 1.5                                                                          22.7 ± 1.8                                          0.0001        20.8 ± 0.2                                                                          23.1 ± 2.1                                          (OKT4.sup.+ OKT8.sup.+)                                                       1             1.2      1.9*                                                   0.01          1.2      1.8*                                                   0.0001        1.2      1.1                                                    ______________________________________                                         *statistically significant at the p < 0.05 level when compared with the       control values.                                                          

The results presented in TABLE 52 show L--Glu--L--Trp increased thepercentage of detectable T-helpers at concentrations of 1 and 0.01mg/mL. L--Glu--L--Trp did not alter the percentage of detectable OKT 8⁺T-suppressor cells.

EXAMPLE 29 Effects of Dipeptide L--Glu--L--Trp on Immunity inExperimental Animals

Protocol A: Healthy Guinea Pigs

Immune parameters were investigated in healthy male guinea pigs(250-300×g) following administration of test or control agents. Eachtest and control group consisted of 10 animals. Test agents wereadministered to animals once daily by the intramuscular (im) route overa period of 5 days and at 1 mg/kg dipeptide L--Glu--L--Trp.Physiological saline was administered im to the animals of the controlgroup. The affects of these treatments were determined on day 10 (i.e.,5 days after the last injection) by preparing cells from peripheralblood (PBL), thymus (TYM), lymph nodes (LN), spleen (SPL), and red pulpof bone marrow (BM). Cells were tested for antibody Fc receptors(EA-RFC), complement receptors (EAC-RFC), T-lymphocytes, "active"T-lymphocytes (E-RFC), B-lymphocytes (EA-RFC and EAC-RFC; according tothe method of Bianco, C., et al., J. Exp. Med. 132(4):702-720 (1970).The results of these analyses are shown in TABLES 53-54, below, wheredata are expressed either as the number of RFC×10⁹ per liter of blood,or RFC×10³ per milligram (mg) of tissue.

                                      TABLE 53                                    __________________________________________________________________________    Effect of L--Glu--L--Trp on                                                   Immune Indices: E-RFC (X ± S.D.)                                                Index                                                                    Cell per E-RFC (X ± S.D.)                                                                         EA-RFC (X ± S.D.)                                   Population                                                                         10.sup.x cells                                                                    Control                                                                             L--Glu--L--Trp                                                                        Control                                                                             L--Glu--L--Trp                                   __________________________________________________________________________    PBL  10.sup.9 /L                                                                       0.53 ± 0.10                                                                      1.59 ± 0.78                                                                        0.50 ± 0.09                                                                      0.82 ± 0.09                                   TYM  10.sup.3 /mg                                                                      440.4 ± 82.3                                                                     448.1 ± 51.4                                                                       345.6 ± 63.2                                                                     542.6 ± 42.3                                  LN   10.sup.3 /mg                                                                      119.1 ± 19.3                                                                     81.4 ± 7.6*                                                                        787 ± 8.3                                                                        61.7 ± 8.2                                    SPL  10.sup.3 /mg                                                                      69.5 ± 6.6                                                                       75.9 ± 6.8                                                                         48.5 ± 6.4                                                                       81.2 ± 7.4*                                   BM   10.sup.3 /mg                                                                      22.5 ± 3.9                                                                       11.2 ± 2.3*                                                                        19.1 ± 2.2                                                                       25.1 ± 1.9*                                   __________________________________________________________________________     *statistically significant at the p < 0.05 level in comparison with the       indices in the control.                                                  

                  TABLE 54                                                        ______________________________________                                        Effect of L--Glu--L--Trp on                                                   Immune Indices: EAC-RFC (X ± S.D.)                                         Cell     Index per                                                            Population                                                                             10.sup.x cells                                                                           Control      L--Glu--L--Trp                               ______________________________________                                        PBL      10.sup.9 /L                                                                              0.30 ± 0.09                                                                             0.79 ± 0.08*                              TYM      10.sup.3 /mg                                                                             3.4 ± 0.7 0                                            LN       10.sup.3 /mg                                                                             146.5 ± 12.4                                                                            173.3 ± 12.7                              SPL      10.sup.3 /mg                                                                             140.2 ± 15.6                                                                            141.5 ± 22.6                              BM       10.sup.3 /mg                                                                             19.7 ± 3.2                                                                              13.9 ± 2.1                                ______________________________________                                         *statistically significant at the p < 0.05 level in comparison with the       indices in the control.                                                  

The results presented in TABLES 53-54 show that animals treated for 5days with the dipeptide L--Glu--L--Trp had 3-fold more T-lymphocytes inperipheral blood (i.e., PBL; E-RFC) than control animals treated withsaline. T-lymphocytes in LN and BM decreased (i.e., 50%) while splenicand thymus T-cells increased 2-9% after L--Glu--L--Trp treatment. At 10days, animals who received L--Glu--L--Trp showed an increased number ofT-lymphocytes in peripheral blood, spleen, and bone marrow.

L--Glu--L--Trp increased Fc-receptor bearing cells in spleen and bonemarrow (TABLE 53). Numbers of complement receptor bearing cells(EAC-RFC) were increased 2-fold in blood by L--Glu--L--Trp treatment(TABLE 54).

The results indicate that L--Glu--L--Trp stimulated proliferation,differentiation, or migration of T- and B-lymphocytes.

Protocol B: Gamma-Irradiated Guinea Pigs

X-irradiation an accepted experimental animal model for depressingcellular immune mechanisms operative in anti-bacterial, anti-viral, andanti-parasitic immunity. The levels of lymphocytes in thymus, spleen,and lymph node are decreased following sub-lethal radiation exposure,and the effects of L--Glu--L--Trp on recovery of immune functionfollowing radiation exposure were investigated in this model.

142 guinea pigs were exposed to 1 Gy of X-irradiation and then treatedwith L--Glu--L--Trp at a dose of 0.01 mg/kg daily for 5 days startingtwo days after the irradiation. Immunologic indices were determined at5, 10, and 20 days by preparing samples of immune cells from thymus,spleen, lymph nodes, and peripheral blood. Cell preparations wereanalyzed to determine the content of lymphocytes, E-RFC (i.e.,T-lymphocytes), EA-RFC ("activated" T-lymphocytes), and EAC-RFC (i.e.,B-lymphocytes). (The materials and methods used in these analyses arepresented following EXAMPLE 38, below. The results obtained in theseexperiments are summarized in TABLE 55.

                                      TABLE 55                                    __________________________________________________________________________    Effect of L--Glu--L--Trp on Recovery of Immune Cells                          in Irradiated Guinea Pigs (X ± S.D.): Thymic Lymphocytes                          Lymphocytes × 10.sup.9 /L (% Normal).sup.a                              Normal   Irradiated                                                                             Irradiated and                                       Cell   Healthy  Un-treated                                                                             L--Glu--L--Trp                                       Population                                                                           Control I                                                                              Control II                                                                             Treated                                              __________________________________________________________________________    5 Days Post Irradiation                                                       __________________________________________________________________________    Lymphocytes                                                                          481+/-21                                                                           (100%)                                                                            270+/-32*                                                                          (56%)                                                                             395+/-27**                                                                          (82%)                                          E-RFC  378+/-26                                                                           (100%)                                                                            93+/-10*                                                                           (25%)                                                                             252+/-26**                                                                          (67%)                                          EA-RFC 290+/-28                                                                           (100%)                                                                            74+/-8*                                                                            (26%)                                                                             150+/-16**                                                                          (52%)                                          EAC-RFC                                                                              530+/-0.3                                                                          (100%)                                                                            7+/-1*                                                                             (140%)                                                                            4+/-1**                                                                             (80%)                                          __________________________________________________________________________    10 Days Post Irradiation                                                      __________________________________________________________________________    Lymphocytes                                                                          481+/-21                                                                           (100%)                                                                            87+/-13*                                                                           (18%)                                                                             276+/-26**                                                                          (57%)                                          E-RFC  379+/-26                                                                           (100%)                                                                            50+/-6*                                                                            (13%)                                                                             195+/-21**                                                                          (51%)                                          EA-RFC 290+/-28                                                                           (100%)                                                                            31+/-3*                                                                            (11%)                                                                             143+/-16**                                                                          (49%)                                          EAC-RFC                                                                              5+/-0.3                                                                            (100%)                                                                            6+/-1                                                                              (120%)                                                                            3+/-0.4*                                                                            (60%)                                          __________________________________________________________________________    20 Days Post Irradiation                                                      __________________________________________________________________________    Lymphocytes                                                                          481+/-21 254+/-33*                                                                              423+/-40**                                           E-RFC  (100%)   (53%)    (88%)                                                EA-RFC 379+/-26 71+/-6*  343+/-30**                                           EAC-RFC                                                                              (100%)   (19%)    (91%)                                                       290+/-28 106+/-13*                                                                              243+/-28**                                                  (100%)   (37%)    (84%)                                                       5+/-0.3  4+/-0.5  4+/-0.4                                                     (100%)   (80%)    (80%)                                                __________________________________________________________________________     .sup.a Cell counts × 10.sup.9 /L; *, statistically significant at       the p < 0.05 level when compared with Control I; **significant at the p <     0.05 level (compared with Control II).                                   

The results presented in TABLE 55 show a drop in thymic lymphocytecounts in irradiated control animals (i.e., Control II), i.e., at 5 daysand 10 the lymphocyte counts decreased to 56% and then 18%,respectively, of normal levels. A partial return of immune cells wasobserved at day 20 with 53% of normal lymphocyte levels. Lymphocytesubpopulations in irradiated control decreased in a parallel fashion,i.e., both T- and B-lymphocytes. L--Glu--L--Trp treatments resulted in astatistically significant increase in the number of thymic lymphocytes,i.e., from 56% to.82% of normal, on day 5; and from 18% to 56% on day10. Similarly, thymic T-lymphocyte counts were increased on day 5 from25% to 67% and on day 10 from 13% to 51%. L--Glu--L--Trp treatment wasfound to exhibit similar effects on splenic and lymph node T- andB-lymphocytes, TABLE 56.

                                      TABLE 56                                    __________________________________________________________________________    Effect of L--Glu--L--Trp on Recovery of Immune Cells                          in Irradiated Guinea Pigs (X ± S.D.): Splenic Lymphocytes                         Lymphocytes × 10.sup.9 /L (% Normal).sup.a                              Normal   Irradiated                                                                             Irradiated and                                       Cell   Healthy  Un-treated                                                                             L--Glu--L--Trp                                       Population                                                                           Control I                                                                              Control II                                                                             Treated                                              __________________________________________________________________________    5 Days Post Irradiation                                                       __________________________________________________________________________    Lymphocytes                                                                          409+/-26                                                                           (100%)                                                                            273+/-32*                                                                          (67%)                                                                             378+/-36**                                                                          (92%)                                          E-RFC  40+/-3                                                                             (100%)                                                                            93+/-10*                                                                           (230%)                                                                            48+/-4**                                                                            (120%)                                         EA-RFC 26+/-2                                                                             (100%)                                                                            8+/-1*                                                                             (31%)                                                                             22+/-4**                                                                            (85%)                                          EAC-RFC                                                                              73+/-5                                                                             (100%)                                                                            36+/-3*                                                                            (49%)                                                                             60+/-5**                                                                            (82%)                                          __________________________________________________________________________    10 Days Post Irradiation                                                      __________________________________________________________________________    Lymphocytes                                                                          409+/-26                                                                           (100%)                                                                            148+/-23*                                                                          (36%)                                                                             147+/-18**                                                                          (36%)                                          E-RFC  40+/-3                                                                             (100%)                                                                            30+/-3*                                                                            (75%)                                                                             29+/-3*                                                                             (73%)                                          EA-RFC 26+/-2                                                                             (100%)                                                                            15+/-2*                                                                            (58%)                                                                             15+/-2*                                                                             (58%)                                          EAC-RFC                                                                              73+/-5                                                                             (100%)                                                                            51+/-4*                                                                            (70%)                                                                             73+/-5**                                                                            (100%)                                         __________________________________________________________________________    20 Days Post Irradiation                                                      __________________________________________________________________________    Lymphocytes                                                                          409+/-26                                                                           (100%)                                                                            273+/-47*                                                                          (67%)                                                                             260+/-32**                                                                          (64%)                                          E-RFC  40+/-3                                                                             (100%)                                                                            30+/-3*                                                                            (75%)                                                                             45+/-4**                                                                            (113%)                                         EA-RFC 26+/-2                                                                             (100%)                                                                            20+/-3*                                                                            (77%)                                                                             29+/-4**                                                                            (112%)                                         EAC-RFC                                                                              73+/-5                                                                             (100%)                                                                            67+/-8                                                                             (92%)                                                                             82+/-9                                                                              (112%)                                         __________________________________________________________________________     .sup.a Cell counts × 10.sup.9 /L; *, statistically significant at       the p < 0.05 level when compared with Control I; **significant at the p <     0.05 level (compared with Control II).                                   

The results presented in TABLE 56 show a decrease in the number ofsplenic lymphocytes in irradiated control guinea pigs, dropping to 36%of normal levels on day 10. Predictably, the majority of the decreasewas evident in the short-lived B-lymphocyte subpopulation, and splenicT-lymphocytes proved more radiation resistant. Treatment withL--Glu--L--Trp significantly increased the numbers of splenicB-lymphocytes (i.e., EAC-RFC) on days 5 and 10; with increases on day 5from 49% to 82% of normal, and on day 10 from 70% to 100%.

                                      TABLE 57                                    __________________________________________________________________________    Effect of L--Glu--L--Trp on Recovery of Immune Cells                          in Irradiated Guinea Pigs (X ± S.D.): Lymph Node Lymphocytes                      Lymphocytes × 10.sup.9 /L (% Normal).sup.a                              Normal   Irradiated                                                                             Irradiated and                                       Cell   Healthy  Un-treated                                                                             L--Glu--L--Trp                                       Population                                                                           Control I                                                                              Control II                                                                             Treated                                              __________________________________________________________________________    5 Days Post Irradiation                                                       __________________________________________________________________________    Lymphocytes                                                                          252+/-18                                                                           (100%)                                                                            171+/-22*                                                                          (68%)                                                                             183+/-21*                                                                           (73%)                                          E-RFC  33+/-2                                                                             (100%)                                                                            24+/-3*                                                                            (73%)                                                                             40+/-4**                                                                            (122%)                                         EA-RFC 18+/-1                                                                             (100%)                                                                            11+/-2                                                                             (61%)                                                                             20+/-3**                                                                            (111%)                                         EAC-RFC                                                                              56+/-4                                                                             (100%)                                                                            9+/-1*                                                                             (16%)                                                                             25+/-2**                                                                            (45%)                                          __________________________________________________________________________    10 Days Post Irradiation                                                      __________________________________________________________________________    Lymphocytes                                                                          252+/-18                                                                           (100%)                                                                            57+/-11*                                                                           (23%)                                                                             75+/-13*                                                                            (30%)                                          E-RFC  33+/-2                                                                             (100%)                                                                            15+/-2*                                                                            (45%)                                                                             29+/-3**                                                                            (88%)                                          EA-RFC 18+/-1                                                                             (100%)                                                                            10+/-1*                                                                            (56%)                                                                             17+/-2**                                                                            (94%)                                          EAC-RFC                                                                              56+/-4                                                                             (100%)                                                                            8+/-1*                                                                             (14%)                                                                             12+/-1*                                                                             (21%)                                          __________________________________________________________________________    20 Days Post Irradiation                                                      __________________________________________________________________________    Lymphocytes                                                                          252+/-18                                                                           (100%)                                                                            109+/-47*                                                                          (67%)                                                                             242+/-31**                                                                          (96%)                                          E-RFC  33+/-2                                                                             (100%)                                                                            18+/-2*                                                                            (75%)                                                                             30+/-2**                                                                            (91%)                                          EA-RFC 18+/-1                                                                             (100%)                                                                            9+/-1*                                                                             (50%)                                                                             25+/-4**                                                                            (1399)                                         EAC-RFC                                                                              56+/-4                                                                             (100%)                                                                            8+/-1*                                                                             (14%)                                                                             9+/-1*                                                                              (16%)                                          __________________________________________________________________________     .sup.a Cell counts × 10.sup.9 /L; *, statistically significant at       the p < 0.05 level when compared with Control I; **significant at the p <     0.05 level (compared with Control II).                                   

The results presented in TABLE 57 show that irradiated guinea pigsexhibited decreased numbers of T- and B-lymphocytes in lymph nodes,i.e., 23% of normal T-cell levels on day 10 and only 14% of normalB-cell levels. Treatment with L--Glu--L--Trp significantly increasedT-lymphocyte counts in lymph nodes on days 5, 10 and 20 and B-lymphocytecounts were statistically elevated on day 5. On day 10 and day 20 B-cellcounts in the lymph nodes of L--Glu--L--Trp treated animals remainedlow, presumably because afferent repopulation with immune cells requiresantigen stimulation.

                  TABLE 58                                                        ______________________________________                                        Effect of L--Glu--L--Trp on Recovery of Functional Activity of                Immune Cells in the Peripheral Blood of Irradiated Guinea Pigs                (X ± S.D.)                                                                          Normal     Irradiated Irradiated and                                 Days     Healthy    Un-Treated L--Glu--L--Trp                                 Post-Irradiation                                                                       Control I  Control II Treated                                        ______________________________________                                        Lymphocyte Blastogenesis with Con-A (%)                                       ______________________________________                                         5       36+/-1     66+/-4*    44+/-4**                                       10       37+/-2     81+/-7*    67+/-5**                                       20       37+/-2     76+/-7*    49+/-2**                                       ______________________________________                                        Neutrophil Cationic Protein Levels                                            ______________________________________                                         5       1.79+/-0.07                                                                              1.36+/-0.09*                                                                             1.42+/-0.15*                                   10       1.76+/-0.07                                                                              1.29+/-0.08*                                                                             1.54+/-0.09**                                  20       1.81+/-0.08                                                                              1.47+/-0.12*                                                                             1.63+/-0.11**                                  ______________________________________                                         *p < 0.05 in comparison with Control I; **p < 0.05 in comparison with         Control II.                                                              

As expected, the results presented in TABLE 58 show that peripheralblood lymphocytes from irradiated guinea pigs exhibited increasednon-specific radiation-induced responsive to Concanavalin-A mitogen, anddecreased levels of neutrophil cationic proteins. (Increasednon-specific mitogen responsiveness in irradiated animals is correlatedwith decreased lymphocyte responsiveness to specific antigens, e.g.,pathogens.) Treatment with L--Glu--L--Trp resulted in a statisticallysignificant decrease in Con-A responsiveness and increased levels ofneutrophil cationic proteins. Both of these findings are consistent withthe conclusion that L--Glu--L--Trp treatments increasedneutrophil-mediated immunity to pathogens, and lymphocyte responsivenessto foreign agents.

EXAMPLE 30 Effects of L--Glu--L--Trp Dipeptide on Indices of InnateImmunity

The effects of L--Glu--L--Trp on innate mechanisms of immunity wereinvestigated in male CBA mice (18-20 g). Eighty mice were divided into 4groups of 20 mice each. The animals in each of the groups were injectedintraperitoneally (ip) once daily over a period of 6 days with either:i) 1 mg/kg L--Glu--L--Trp, ii) 0.01 mg/kg L--Glu--L--Trp, or iii)non-pyrogenic physiological saline. Next (on day 6), each group ofanimals was sub-divided into two subgroups of 10 animals each: i.e., onesubgroup of animals was injected ip with 10% sterile proteose peptone toinduce neutrophils (induced), while the other subgroup were non-induced(non-induced). The animals of the four induced-subgroups were sacrificed2.5 hours after the ip injection. Thymus and spleen weights weredetermined in the non-induced animals; cell suspensions were preparedfrom each thymus and spleen; and, resident peritoneal exudate cells(PEC) were harvested by lavage with Medium 199 from each animal. Thepercentage content of T- and B-lymphocytes in the splenic cellpopulations from the non-induced animals was determined byphase-contrast and indirect immunofluorescence Shtorkh, V., & Emmrikh,I. M., Immunological methods, Medicine, Russia. pp. 254-268 (1987))using rabbit antisera specific for murine Thy-1 and immunoglobulin.(Rabbit anti-Thy-1 was raised by immunization with a murine brainhomogenate (Jolyb, E. S., Cell Immunol. 2:353-361 (197 1)) and anti-Igby immunization with an ammonium sulfate precipitate of murine serum.The percentage macrophages in the resident PEC populations weredetermined in Romanovsky stained smears of cells, the cells thencollected by centrifugation at 150×g/10 minutes, and macrophage activitymeasured by reduction of nitroblue tetrazolium (NBT; see Jolyb, supra)before and after induction with complement-opsonized zymosan (guinea pigcomplement activated by zymosan;). NBT reduction was quantifiedspetrophotometrically at 540 nm. Pinocytic activity of the residentperitoneal macrophages was assessed by measuring uptake of neutral reddye (measured spectrophotometrically). The percentage of neutrophils inthe PEC population was determined using phase contrast microscopy andcomplement-opsonized zymosan. Phagocytic activity of neutrophils wasdetermined by incubating the cells with 2.5×10⁸ Staphylococcus aureusper milliliter (S. aureus from a 24 hour culture). The data wereexpressed as both the percentage of phagocytic cells (% of the totalcell population phagocytizing 1 or more Staphylococci) and thephagocytic index (expressed as the mean number of intracellular bacteriainside each of the phagocytically active neutrophils), both measurementsbeing made by microscopic examination following Romanovsky-Giemsastaining of cell smears.

The results of these respective experimental measurements on cellscontributing to innate mechanisms of immunity are summarized in TABLES59-64.

                                      TABLE 59                                    __________________________________________________________________________    Effect of L--Glu--L--Trp on Thymic and Spleen Mass (X ± S.D.)                      No. Dose                                                                              Thymus                                                                              Thymus                                                                              Spleen                                                                              Splenic                                     Group   Animals                                                                           (mg/kg)                                                                           (mg)  Change (%)†                                                                  (mg)  Change (%)                                  __________________________________________________________________________    Saline Control                                                                        10  0   25.4 ± 1.6                                                                       0     81.7 ± 3.4                                                                       0                                           L--Glu--L--Trp                                                                        10  1.0 14.1 ± 1.3*                                                                      ↓ 44                                                                         88.4 ± 4.4                                                                       ↑ 8                                           10  0.01                                                                              14.8 ± 1.6*                                                                      ↓ 42                                                                         69.8 ± 2.6**                                                                     ↓ 15                                 __________________________________________________________________________     *, statistically significant at the p < 0.01 level when compared with         saline control; **significant at the p < 0.05 level (compared with saline     controls); †, percentage change of the mean value (%) relative to      the mean value of the saline control.                                    

The results presented in TABLE 59 show L--Glu--L--Trp induced astatistically significant decrease in thymic weight at both dosages, andhad opposing effects on splenic weight at the indicated dosages.Interestingly, the 1 mg/kg dosage of L--Glu--L--Trp administered inthese studies approximates a clinically effective dosage in humans forimmune stimulation.

The results presented in TABLES 60-65, show the effects of this agent onindividual cell populations and their activities.

TABLE 60 demonstrates the effects of L--Glu--L--Trp on the meanpercentage of T- and B-lymphocytes isolated in the cell populations fromthe spleens of treated animals

                                      TABLE 60                                    __________________________________________________________________________    Effects of L--Glu--L--Trp on the                                              Percentage of Splenic T- and B-lymphocytes (X ± S.D.)                              Dose ip                                                                           Dose                                                                              Lymphocytes (%)                                               Group   (μg/ml)                                                                        (mg/kg)                                                                           T cells (%)                                                                         Change†                                                                     B cells (%)                                                                         Change                                       __________________________________________________________________________    Saline Control                                                                        0.1 0   37.0 ± 1.28                                                                      0.0  50.0 ± 1.71                                                                      0.0                                          L--Glu--L--Trp                                                                        10  1.0 34.8 ± 2.5                                                                       ↓ 0.9                                                                       52.4 ± 1.71                                                                      1.0                                                  0.1 0.01                                                                              41.2 ± 2.1*                                                                      ↑ 1.1                                                                        50.8 ± 3.0                                                                       1.0                                          __________________________________________________________________________     *, statistically significant at the p < 0.01 level in comparison with the     saline control group results, †, Change = % peptide treated/%          control.                                                                 

The results presented in TABLE 60 show that L--Glu--L--Trp slightlyaltered the percentage of splenic T-lymphocytes. In these studies theB-lymphocyte content of the spleen did not seem to be affected by theagent.

TABLE 61 shows the results of studies with peritoneal macrophages andtheir state of activation as measured by the ability to reduce NBT.

                  TABLE 61                                                        ______________________________________                                        Effects of L--Glu--L--Trp on the                                              Innate State of Activation of Resident Peritoneal Macrophages as              Determined by NBT Reduction                                                           Dose    NBT Reduction (OD.sub.540)*                                   Group     (mg/kg)   Spontaneous Induced†                               ______________________________________                                        Saline Control                                                                          0         0.055 ± 0.002                                                                          0.105 ± 0.005                              L--Glu--L--Trp                                                                          1         0.059 ± 0.003                                                                          0.116 ± 0.008                                        0.01      0.101 ± 0.002**                                                                        0.205 ± 0.012**                            ______________________________________                                         *, mean ± S.D. of four determinations for each group; **, statisticall     significant at the p < 0.05 level from the values recorded in the control     group; †, macrophages induced with complement opsonized zymosan.  

The results presented in TABLE 61 show, i) as expected,complement-opsonized zymosan activated resident peritoneal macrophages(saline controls) by about 1.9-fold; ii) treatment of animals with a sixday ip treatment course of L--Glu--L--Trp (0.01 mg/kg) resulted in aspontaneous level of macrophage activity that approximated the activityobserved with the induced resident peritoneal macrophages (i.e., in thesaline treated controls); iii) when macrophages from L--Glu--L--Trp(0.01 mg/kg) treated animals were induced with complement-opsonizedzymosan, 1.75-fold and 2-fold increases, respectively, in NBT reducingactivity were observed

In TABLE 62 are shown the results of experiments designed to examine thepinocytic activity of resident peritoneal macrophages from therespective treatment groups.

                  TABLE 62                                                        ______________________________________                                        Effect of L--Glu--L--Trp on Uptake of Neutral Red                             by Resident Peritoneal Macrophages (X ± S.D.)†                              Dose    Neutral Red Uptake                                            Group     (mg/kg)   No. of Assays                                                                            Absorbance                                     ______________________________________                                        Saline Control                                                                          0         5          0.488 ± 0.026                               L--Glu--L--Trp                                                                          1         5          0.469 ± 0.016                                         0.01      4          0.604 ± 0.031**                             ______________________________________                                         †, mean absorbance values ± standard deviation of the mean;         *,statistically significant at the p < 0.01 level in comparison with the      control values; **, p < 0.05                                             

The results presented in TABLE 62 show a 75% increase in neutral reduptake by macrophages from L--Glu--L--Trp treated animals (0.01 mg/kg),however, this difference was not statistically different from thecontrol values.

TABLE 63 shows the results of experiments designed to investigate thetissue response of neutrophils to an inflammatory agent, i.e., inducedin the peritoneum by injection of sterile proteose peptone.

                  TABLE 63                                                        ______________________________________                                        Effects of L--Glu--L--Trp Intraperitoneal Pretreatment                        on Neutrophil Response to Proteose Peptone (X ± S.D.)                                 Dose ip Dose    Total Number of Cells Induced                      Group      (μg/ml)                                                                            (mg/kg) (× 10.sup.6)                                 ______________________________________                                        Saline Control                                                                           0.1     0       40                                                 L--Glu--L--Trp                                                                           10      1        80*                                                          0.1     0.01    50                                                 ______________________________________                                         *,statistically significant at the p < 0.05 level compared with values        recorded in the control group.                                           

The results presented in TABLE 63 show that ip pretreatment withL--Glu--L--Trp (0.1 mg/kg) doubled the number of neutrophils that couldbe induced following injection of proteose peptone.

It is interesting that the total volume of exudate obtained fromL--Glu--L--Trp treated animals (0.01 mg/kg) was increased relative tothe volume obtained from controls and the cell concentration wascorrespondingly decreased, i.e., 0.9×10⁶ for L--Glu--L--Trp versus1.9×10⁶ for control.

In TABLE 64, are shown the results of experiments designed to test thephagocytic activity of the neutrophils induced by the proteose peptoneinjection.

                  TABLE 64                                                        ______________________________________                                        Effect of L--Glu--L--Trp Pretreatment on                                      Phagocytic Activity of Neutrophils Responding to Induction                                      Phagocytic Activity                                                                             Phagocytic                                                                    Index                                               Dose ip  Dose     Phagocytes                                                                            (No. bacteria/                            Group     (μg/ml)                                                                             (mg/kg)  (%)     phagocyte)                                ______________________________________                                        Saline Control                                                                           0       0        18.8 ± 0.29                                                                        2.05 ± 0.10                            L--Glu--L--Trp                                                                          10       1        31.5 ± 0.4*                                                                        1.89 ± 0.03                                      0.1      0.1      17.8 ± 1.13                                                                        1.90 ± 0.05                            ______________________________________                                         *, statistically significant at the p < 0.01 level in comparison with the     values recorded in controls.                                             

The results presented in TABLE 64 show that the six day ip pretreatmentcourse of L--Glu--L--Trp (1 mg/kg) induced statistically significant1.5-fold and 1.7-fold increases, respectively, in the percentage ofphagocytic cells induced into the peritoneal cavity following injectionof a sterile inflammatory challenge with proteose peptone. Thephagocytic capacity of the neutrophils induced in this manner wasfinite, i.e., about two Staphylococci per neutrophil in saline controlor peptide treated animals. L--Glu--L--Trp pretreatment at a dose of0.01 mg/kg did not appear to stimulate neutrophil migration in responseto the sterile proteose peptone inflammatory stimulus (TABLE 63), northe phagocytic activity of the cells so induced (TABLE 64).

The results presented in EXAMPLES 26-29, above, indicate the following:i) L--Glu--L--Trp treatment can cause redistribution of lymphoid cellpopulations in animals; ii) L--Glu--L--Trp treatment did not alter theactivity of resident peritoneal macrophages; iii) L--Glu--L--Trptreatment stimulated both the number of neutrophils entering the tissuein response to a sterile inflammatory challenge and the percentage ofphagocytically active cells in the cell exudate population; iv)L--Glu--L--Trp appeared to exhibit dosage-dependent differentialeffects, i.e., at the 1 mg/kg dose neutrophils were stimulated, but atthe lower 0.01 mg/kg dose, macrophages, and not neutrophils, werestimulated, with a possible mobilization of thymic and bone marrowlymphocytes and increased T-lymphocytes in the spleen at the conclusionof the 6 day ip treatment regimen.

The combined results indicate that treatment with L--Glu--L--Trp caninduce changes in distribution of lymphocyte cell populations, andchanges in distribution and activities of macrophages and neutrophils,both of which changes favor a heightened state of innate immunity in thetreated animals. For these and other reasons L--Glu--L--Trp is termed an"immunomodulator".

EXAMPLE 31 Effects of L--Glu--L--Trp on Induction of Antibody Responsesas Measured by Jerne Plaque Formation

Thirteen male (CBAxC57B1/6) F1 mice, 10-12 weeks of age (22±0.2 g) werechallenged with a single intraperitoneal (ip) injection of 2.5×10⁷ sheepred blood cells (s-rbc) in 0.5 mL normal saline. Within 20 minutes afterthe injection of s-rbc an L--Glu--L--Trp treatment trial was initiatedby injecting 0.2 mL of the dipeptide preparation at a concentrationsufficient to deliver a unit dosage of 1.0 μg/kg, 10 μg/kg, or 100μg/kg. The same dosage was delivered daily for the next three days.Control animals (13 per group) included: i) non-s-rbc-immunized and ii)s-rbc immunized but non L--Glu--L--Trp-treated. At day 5 (post-s-rbcchallenge), the spleens of the animals were removed and splenocytesprepared by homogenizing the spleens in a loose fitting glassDounce-type homogenizer, followed by screening of the cell suspensionthrough a caprone filter, and differential centrifugation (yield about10⁸ cells/spleen).

Antibody-producing cells (AbPC) in the splenocyte preparations werequantified using the well known method of Jerne and Nordin. Briefly,concentrations of 5×10⁴ to 10⁶ splenocytes were mixed with a 2% solutionof s-rbc (% volume packed cells/volume medium) and incorporated into anagarose layer in petri dishes. The dishes were incubated for 60 minutesat 37° C. (in a humidified incubator in an atmosphere of 5% CO₂), toallow APC to synthesize antibody capable of binding to the surroundings-rbc in the agarose matrix. After the 60 minute incubation, a 1:20dilution of guinea pig serum was added as a source of complement andclear zones of s-rbc lysis (surrounding the AbPCs) were recorded bymicroscopic observation after an additional 60 minutes incubation. Theresults are summarized in TABLE 65.

                                      TABLE 65                                    __________________________________________________________________________    Effects of L--Glu--L--Trp on the Antibody Plaque Forming                      Cell Response of Mice to Sheep Red Blood Cells (X ± S.D.)                                     Splenocytes                                                Test    Dose                                                                              Spleen weight/                                                                       per            PFC/10.sup.6                                Agent   (μg/kg)                                                                        Body weight                                                                          Spleen (× 10.sup.6)                                                            PFC/Spleen                                                                            splenocytes                                 __________________________________________________________________________    None    0   0.44 ± 0.02                                                                       159.5 ± 0.1                                                                       80 ± 19                                                                            0.5 ± 0.1                                Saline  0   0.53 ± 0.05                                                                       184.2 ± 7.3*                                                                      4284 ± 579*                                                                        24.3 ± 4.2*                              control                                                                       L--Glu--L--Trp                                                                        1   0.42 ± 0.01*                                                                      168.6 ± 3.6                                                                       11722 ± 2005*†                                                              69.2 ± 11.3*†                             10  0.43 ± 0.01                                                                       159 ± 8.5†                                                                 7854 ± 1631*                                                                       48.8 ± 9*†                                100 0.44 ± 0.02                                                                       162.6 ± 6†                                                                 6731 ± 1027*                                                                       41.7 ± 6*†                        __________________________________________________________________________     *statistically significant at the p < 0.05 level (Student's ttest)            relative to the values recorded in the Nonimmunized control group of          animals;                                                                      †statistically significant at the p < 0.05 level relative to the       values recorded in the srbc immunized, saline treated, control group of       animals.                                                                 

The results presented in TABLE 65 show that 3 daily ip treatments withL--Glu--L--Trp at a dose of 1 μg/kg, when delivered after an ipchallenge with s-rbc, significantly increased the number of PFC inspleen by 2.7-fold over the values recorded in the saline-treatedcontrol animals. The frequency of PFC's expressed per 10⁶ splenocytesalso increased by 2.8-fold. Spleen weights of the animals treated withthe 1 μg/kg dosage of L--Glu--L--Trp did not increase, and in fact,decreased slightly (but significantly), and total splenocytes harvestedfrom the treated animals were also lower, but still within the rangeharvested from the saline control animals. Thus, treatment with 1 μg/kgL--Glu--L--Trp increased the number and frequency of antigen producingcells within the total spleen cell population but without increasing thetotal number of cells in the spleen. Interestingly, treatments atdosages of 10 μg/kg and 100 μg/kg L--Glu--L--Trp resulted insignificantly fewer splenocytes being harvested than from the salinecontrol animals, and although 1.8-fold and 1.6-fold more PFCs wererecorded (on a per spleen basis) than in saline control animals, thepossibility exists that the higher doses activated AbPCs at the localsite (or in local lymph nodes), and that the cells so activated failedto traffic to the spleen.

EXAMPLE 32 Therapeutic Efficacy of L--Glu--L--Trp in an ExperimentalAnimal Model of Acute Peritoneal Bacterial Infection as Determined bySurvival Following Intraperitoneal Infection with an LD₁₀₀ dose of E.coli

Therapeutic efficacy of L--Glu--L--Trp was tested in a murineexperimental animal model of acute gram negative infection. The lethaldosage of E. coli gram negative rods in the experimental model was in astepwise manner. First, the number of bacteria equivalent to the LD₅₀dose (50% survival) was determined by injecting intraperitoneallydifferent log dosages of bacteria and determining the percentage ofanimals surviving 72 hours later. The LD₁₀₀ dose (100% mortality) wascalculated by multiplying the number of bacteria in the LD₅₀ dose times10. Second, the mean effective dose (MED) of ampicillin sufficient toeffect 100% survival of animals receiving one LD₅₀ dose ofantibiotic-sensitive bacteria was determined.

Mice were divided into five L--Glu--L--Trp treatment groups, fivecombined treatment groups (i.e., L--Glu--L--Trp+antibiotic), anantibiotic treatment control group (Ampicillin), and a saline controlgroup (Control), with eighteen animals in each group. In theL--Glu--L--Trp treatment groups, mice received a prophylacticL--Glu--L--Trp treatment administered daily beginning 3 days beforebacterial challenge (i.e., day 3 through day 1) as intraperitonealinjections of 0.01 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 10 μg/kg, or 100 μg/kg.In the five combined treatment groups, mice received the same fiveintraperitoneal dosages of L--Glu--L--Trp and, in addition, ampicillinequivalent to one MED. In the antibiotic control group, mice receivedonly ampicillin, and in the saline control group, an injection of salineadministered under the same conditions.

On day 0, a number of bacteria equivalent to one LD₁₀₀ was administeredto all animals. Survival was determined after 24, 48, and 72 hours. Theresults of these studies are summarized in TABLE 66.

                  TABLE 66                                                        ______________________________________                                        Effects of L--Glu--L--Trp on Survival Rate (%) of Mice as a                   Function of Time After Induction of Acute Peritonitis by Injection            of E. Coli                                                                              Glu--Trp                                                                              Percentage Survivors:                                                 Dose    Hours After Injection                                       Group       (μg/kg)                                                                              24       48     72                                      ______________________________________                                        Saline Control                                                                            0         0        0      0                                       L--Glu--L--Trp                                                                            10        83.3*    11.1   11.1                                                100       77.8*†                                                                          38.9*  38.9*                                               1000      83.3*†                                                                          44.4*  44.4*                                   Ampicillin Control                                                                        1 MED     33.3*    22.2*  22.2*                                   L--Glu--L--Trp                                                                            10        94.4*†                                                                          44.4*‡                                                                    44.4*‡                       +           100       94.4*†                                                                          66.7*  61.1*†                           Ampicillin  1000      100.0*†                                                                         77.8*‡                                                                    77.8*†‡               ______________________________________                                         *statistically significant at the p < 0.05 level in comparison with the       saline control values; †statistically significant as compared to       the Ampicillintreatment control; ‡statistically significant (p     < 0.05) compared with the corresponding dosage of L--Glu--L--Trp without      Ampicillin.                                                              

The Materials and Methods used in these studies appear immediatelyfollowing EXAMPLE 46.

EXAMPLE 33 Therapeutic Efficacy of L--Glu--L--Trp in an ExperimentalAnimal Model of Acute Peritoneal Bacterial Infection as Determined bySurvival Following Intraperitoneal Infection with an LD₁₀₀ dose ofPseudomonas aeruginosa

Therapeutic efficacy of L--Glu--L--Trp was tested in a murineexperimental animal model of acute gram negative infection. The lethaldosage of Pseudomonas bacilli in the experimental model was in astepwise manner. First, the number of bacilli equivalent to the LD₅₀dose (50% survival) was determined by injecting intraperitoneallydifferent log dosages of bacilli and determining the percentage ofanimals surviving 96 hours later. The theoretical LD₁₀₀ dose (100%mortality) was calculated by multiplying the number of bacteria in theLD₅₀ dose times 10. Second, the mean effective dose (MED) of ampicillinsufficient to effect 100% survival of animals receiving one LD₅₀ dose ofantibiotic-sensitive bacilli was determined.

Mice were divided into five L--Glu--L--Trp treatment groups, fivecombined treatment groups (i.e., L--Glu--L--Trp+antibiotic), anantibiotic treatment control group (Gentamycin), and a saline controlgroup (Control), with eighteen animals in each group. In theL--Glu--L--Trp treatment groups, mice received a prophylacticL--Glu--L--Trp treatment administered daily for 3 days before bacterialchallenge as a daily intraperitoneal injection of 0.01 μg/kg, 0.1 μg/kg,1.0 μg/kg, 10 μg/kg, or 100 μg/kg. In the five combined treatmentgroups, mice received the same five intraperitoneal dosages ofL--Glu--L--Trp and, in addition, gentamycin equivalent to one MED. Inthe antibiotic control group, mice received only gentamycin, and in thesaline control group, an injection of saline administered under the sameconditions. On day 0, a number of bacilli equivalent to one LD₁₀₀ wasadministered to all animals. Survival was determined after 24, 48, and96 hours. The results of these studies are summarized in TABLE 67.

                  TABLE 67                                                        ______________________________________                                        Effects of L--Glu--L--Trp on Survival Rate (%)                                of Mice as a Function of Time After Induction of                              Acute Peritonitis by Injection of Pseudomonas aeruginosa                                Glu--Trp                                                                              Percentage Survivors:                                                 Dose    Hours After Injection                                       Group       (μg/kg)                                                                              24       48     96                                      ______________________________________                                        Saline Control                                                                            0         0        0      0                                       L--Glu--L--Trp                                                                            10        55.6*    5.6    5.6                                                 100       66.7*†                                                                          38.9*  38.9                                                1000      77.8*†                                                                          44.4*  44.4*                                   Gentamycin Control                                                                        1 MED     27.8*    27.8*  27.8*                                   L--Glu--L--Trp                                                                            10        77.8*†                                                                          61.1*†‡                                                            61.1*†‡               +           100       83.3*†                                                                          77.8*†‡                                                            77.8*†‡               Gentamycin  1000      100*†                                                                           88.9*†‡                                                            778*†‡                ______________________________________                                         *statistically significant at the p < 0.05 level in comparison with the       saline control values; †statistically significant (p < 0.05) as        compared to the Gentamycintreatment control; ‡statistically        significant (p < 0.05) compared with the corresponding dosage of              L--Glu--L--Trp without Gentamycin.                                       

The results presented in TABLE 67 show that L--Glu--L--Trp alone, or incombination therapy with gentamycin, significantly increased thepercentage of mice surviving a lethal challenge of Pseudomonas.

EXAMPLE 34 Therapeutic Efficacy of L--Glu--L--Trp in an ExperimentalAnimal Model of Acute Peritoneal Bacterial Infection as Determined bySurvival Following Intraperitoneal Infection with an LD₁₀₀ dose ofAntibiotic-Resistant Staphylococcus aureus

Therapeutic efficacy of L--Glu--L--Trp was tested in a murineexperimental animal model of acute gram positive infection. The lethaldosage of Staphylococci in the experimental model was in a stepwisemanner. First, the number of bacteria equivalent to an LD₅₀ dose (50%survival) was determined by injecting intraperitoneally different logdosages of Staphylococci and determining the percentage of animalssurviving 72 hours later. The theoretical LD₁₀₀ dose (100% mortality)was calculated by multiplying the number of bacteria in the LD₅₀ dosetimes 10. Second, the mean effective dose (MED) of ampicillin sufficientto effect 100% survival of animals receiving one LD₅₀ dose ofmethicillin-sensitive staphylococci was determined, i.e., 100 mg/kg whenadministered ip one hour after bacterial challenge. (In this animalmodel, methicillin-resistant Staphylococci were used as the bacterialchallenge and ampicillin on its own was not sufficient to preventmortality.)

Mice were divided into five L--Glu--L--Trp treatment groups, fivecombined treatment groups (i.e., L--Glu--L--Trp+antibiotic), anantibiotic treatment control group, and a saline control treatmentgroup, with eighteen animals in each group. In the L--Glu--L--Trptreatment groups, mice received a prophylactic L--Glu--L--Trp treatmentadministered daily for 3 days before bacterial challenge as dailyintraperitoneal injections of 0.01 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 10μg/kg, or 100 μg/kg. In the five combined treatment groups, micereceived the same five intraperitoneal dosages of L--Glu--L--Trp and, inaddition, ampicillin equivalent to one MED. In the antibiotic controlgroup, mice received only ampicillin, and in the saline control group,an injection of saline administered under the same conditions. On day 0,a number of bacteria equivalent to one LD₁₀₀ was administered to allanimals. Survival was determined after 24, 48, and 72 hours. The resultsof these studies are summarized in TABLE 68.

                  TABLE 68                                                        ______________________________________                                        Effects of Intraperitoneal Prophylactic Treatment with                        L--Glu--L--Trp on Survival Rate (%) of Mice as a                              Function of Time After Induction of Acute Peritonitis by                      Injection of Staphylococcus aureus                                                      Glu--Trp                                                                              Percentage Survivors:                                                 Dose    Hours After Injection                                       Group       (μg/kg)                                                                              24        48     72                                     ______________________________________                                        Saline Control                                                                            0         0         0      0                                      L--Glu--L--Trp                                                                            0.01      31.5      3.7    0                                                  0.1       33.3      0      0                                                  1         50.0*     27.8*  5.6                                                10        83.3*     50.0*  33.3*                                              100       88.9*     61.1*  50.0*                                                        83.3*     72.2*  55.6*                                  Ampicillin Control                                                                        1 MED     75*       44.4*  30.6*                                  L--Glu--L--Trp                                                                            0.01      75.0*     44.4*  30.6*                                  +           0.1       100.0*†‡                                                              77.8*†‡                                                            61.1*†‡              Ampicillin  1         100.0*†‡                                                              77.8*†‡                                                            66.7*†‡                          10        100.0*†                                                                          83.3*†‡                                                            66.7*†‡                          10        100.0†                                                                           83.3*†‡                                                            72.2*†‡                          100       94.4*     88.9*†                                                                        88.9*†‡              ______________________________________                                         *statistically significant at the p < 0.05 level in comparison with the       saline control values; †statistically significant (p < 0.05) as        compared to the Ampicillintreatment control; ‡statistically        significant (p < 0.05) compared with the corresponding dosage of              L--Glu--L--Trp without Ampicillin.                                       

The results presented in TABLE 68 are presented graphically in FIGS.3A-3D as a function of the i) time after injection of the bacterialchallenge dose (i.e., hrs.); and, ii) the dose in mg/kg of single agentL--Glu--L--Trp or of combination therapy with L--Glu--L--Trp andampicillin.

The results obtained in expanded dose-response studies of single agentand combined treatment with L--Glu--L--Trp ip and im are presented inFIGS. 3C, 3D, 3E, and 3F. The results confirm and extend those presentedin TABLE 68. L--Glu--L--Trp administered by the ip or im routesincreased the percentage of animals surviving challenge with a lethalnumber of Staphylococcus aureus. The experimental protocol disclosed inEXAMPLE 34 was repeated three times with substantially the same results.One of the three experimental iterations utilized S. aureus (ATCC 33593)to control for possible theoretical variations in the laboratory strainof Staphylococci used in the other studies. No difference was notedbetween the results obtained with ATCC 33593 and the laboratorystaphylococcal strain. Additional studies of protection afforded to miceby different routes of delivery of L--Glu--L--Trp are summarized inTABLE 69.

                  TABLE 69                                                        ______________________________________                                        L--Glu--L--Trp Treatment: Survival Rate (%)                                   as a Function of Time and Route of Delivery                                            Glu--Trp                                                                      Dose    Route of Therapy.sup.a                                       Group      (μg/kg)                                                                              ip     im     sc   in                                    ______________________________________                                        Saline Control                                                                             0       ns     ns     ns   ns                                    L--Glu--L--Trp                                                                             1       *      ns     ns   --                                                 3       *      ns     ns   --                                                10       †                                                                             *      *    --                                                30       †                                                                             *      *    --                                                100      †                                                                             †                                                                             †                                                                           --                                                300      †                                                                             †                                                                             †                                                                           ns                                               1000      †                                                                             †                                                                             †                                                                           *                                                2000      --     --     --   *                                                4000      --     --     --   *                                                8000      --     --     --   ns                                    L--Glu--L--Trp                                                                             1       *      ns     *    --                                    +            3       *      *      ns   --                                    Ampicillin  10       *      ns     ns   --                                                30       *      *      ns   --                                                100      †                                                                             *      *    --                                                300      †                                                                             *      *    *                                                1000      †                                                                             †                                                                             *    *                                                2000      --     --     --   ns                                               4000      --     --     --   ns                                               8000      --     --     --   ns                                    ______________________________________                                         .sup.a "ns", no statistically significant effect on mean survival time or     death rate; *, statistically significant (p < 0.05) effect on mean            survival time; "†", statistically significant (p < 0.05) effect on     death rate; "--", not tested.                                            

The duration of the prophylactic effects of L--Glu--L--Trp wereinvestigated by administering therapy ip or im at doses of 1 μg/kg, 100μg/kg, 100,μg/kg, or 1000 μg/kg (alone or in combination withampicillin) at 120 hrs., 72 hrs., 48 hrs., 24 hrs., or 1 hr. beforestaphylococcal challenge. Survival was evaluated (as above) at 24, 48,and 72 hrs. after challenge with an LD₁₀₀ dose of Stahylococci.Administering L--Glu--L--Trp (alone) as a single injection ip 24 hoursbefore bacterial challenge resulted in greater numbers of survivinganimals compared to animals pretreated at 1, 48, or 72 hrs. Combinationtherapy with L--Glu--L--Trp and ampicillin (ip) was also most effectivewhen administered 24 hours before the bacterial challenge. Whencombination therapy (L--Glu--L--Trp+ampicillin) was administered im,rather than ip, at 120 hrs., 72 hrs., or 48 hrs. before bacterialchallenge, a similar percentage of survival resulted, and this wasgreater than that achieved by administering the prophylactic treatment 1hr before bacterial challenge.

The affects of administering multiple prophylactic doses of single agent(L--Glu--L--Trp) or combination therapy (i.e., ampicillin) wasinvestigated by treating ip (or im) at 72, 48, and 24 hrs. beforebacterial challenge. Survival was assessed (as above) at 24, 48, and 72hrs. after challenge with an LD₁₀₀ dose of Staphylococci. The multipletreatment regimen gave significantly higher (p<0.05) survival rates thana single treatment dose at 72, or 48, or 24 hrs. for either single agentor combined therapy, and multiple injections im resulted in highersurvival that if the injections were administered ip.

The therapeutic efficacy of L--Glu--L--Trp was also investigated in thesame experimental animal model of acute bacterial infection byinitiating intraperitoneal infection in mice with an LD₁₀₀ dose ofStaphylococci, and then initiating treatment 1 hour later with an ipinjection of dipeptide, or dipeptide and antibiotic (i.e., ampicillin).The results of these studies are summarized in TABLE 70.

                  TABLE 70                                                        ______________________________________                                        Effects of Intraperitoneal Therapeutic Treatment with                         L--Glu--L--Trp on Survival Rate (%) of Mice as a                              Function of Time After Induction of Acute Peritonitis                         by Injection of Staphylococcus aureus                                                   Glu--Trp                                                                              Percentage Survivors:                                                 Dose    Hours After Injection                                       Group       (μg/kg)                                                                              24       48     72                                      ______________________________________                                        Saline Control                                                                            0         31.5     3.7    0                                       L--Glu--L--Trp                                                                            0.01      16.7     0      0                                                   0.1       5.6*†                                                                           0†                                                                            0                                                   1         27.8†                                                                           5.6†                                                                          0†                                           10        66.7*    38.9*  16.7*                                               100       66.7*    55.6*  33.3*                                   Ampicillin Control                                                                        1 MED     75.0*    44.4*  30.6*                                   L--Glu--L--Trp +                                                                          10        88.3*    61.1*  33.3*†                           Ampicillin  100       88.9*    61.1*  50.0*†                           ______________________________________                                         *statistically significant at the p < 0.05 level in comparison with the       saline control values; †statistically significant (p < 0.05) as        compared to the Ampicillintreatment control; ‡statistically        significant (p < 0.05) compared with the corresponding dosage of              L--Glu--L--Trp without Ampicillin.                                       

The results presented in TABLE 70 shows a dose-response relationshipbetween the amount of L--Glu--L--Trp administered ip and the survival ofanimals challenged with a lethal dose of Staphylococcus aureus.Combination therapy with L--Glu--L--Trp and antibiotic (i.e.,ampicillin) proved more effective than single agent therapy (i.e.,L--Glu--L--Trp alone), and again exhibited a dose-response relationshipbetween treatment dose and survival.

EXAMPLE 35 Effects of Synthetic Peptide L--Glu--L--Trp on HematopoieticActivity of Bone Marrow

The effects of L--Glu--L--Trp on bone marrow hematopoietic activity wasinvestigated in 5-fluorouracil (5-FU) immunosuppressed CBA mice. Micewere treated with a dose schedule of 5-FU determined empirically to besufficient to decrease the absolute number of bone marrow cells andperipheral blood leukocytes by about 50% on day 10-14. Treatment withL--Glu--L--Trp was initiated on day 10, and 1 μg/kg was administered ipdaily on that day and each of the following 4 days. Bone marrow andperipheral blood cell populations were quantified on day 15.

                                      TABLE 71                                    __________________________________________________________________________    Effects of L--Glu--L--Trp Treatments on Bone Marrow Cells in                  5-FU Immunosuppressed CBA Mice.                                                             Normal                                                                             Negative                                                                            Experimental                                                       Control                                                                            Control                                                                             (5-FU + L--Glu--L--Trp                               Cell Type     (untreated)                                                                        (5-FU only)                                                                         Therapy)                                             __________________________________________________________________________    Myelokaryocytes:                                                              Total Cells × 10.sup.6 /mg:                                                           2.44 1.38  1.41                                                 (% of Normal Control Value):                                                                (100%)                                                                             (57%) (58%)                                                Reticular cells: %:                                                                         0.8  0.6   0.7                                                  Total Cells × 10.sup.3 /mg:                                                           19.6 8.3   9.8                                                  (% of Normal Control Value):                                                                (100%)                                                                             (42%) (50%)                                                Non-differentiated blast cells:%:                                                           2.0  2.3   2.2                                                  Total Cells × 10.sup.3 /mg:                                                           48.7 31.7  31.0                                                 (% of Normal Control Value):                                                                (100%)                                                                             (65%) (64%)                                                Myeloblasts: %:                                                                             3.9  6.7   5.0                                                  Total Cells × 10.sup.3 /mg:                                                           95.3 92.4  70.6                                                 (% of Normal Control Value):                                                                (100%)                                                                             (97%) (74%)                                                Promyeloblasts: %:                                                                          2.8  4.9   3.6                                                  Total Cells × 10.sup.3 /mg:                                                           68.3 67.6  50.8                                                 (% of Normal Control Value):                                                                (100%)                                                                             (99%) (74%)                                                Myelocytes + Neutrophil                                                       Metamyelocytes: %:                                                                          5.8  3.4   4.0                                                  Total Cells × 10.sup.3 /mg:                                                           141.5                                                                              46.9  56.4                                                 (% of Normal Control Value):                                                                (100%)                                                                             (33%) (40%)                                                Stab + Seg. neutrophils: %:                                                                 41.9 13.1  24.9                                                 Total Cells × 10.sup.3 /mg:                                                           1022.4                                                                             180.8 351.0                                                (% of Normal Control Value):                                                                (100%)                                                                             (18%) (34%)                                                Eosinophils: %:                                                                             1.0  1.4   1.1                                                  Total Cells × 10.sup.3 /mg:                                                           24.4 19.3  15.6                                                 (% of Normal Control Value):                                                                (100%)                                                                             (79%) (64%)                                                Lymphocytes: %:                                                                             28.8 52.5  43.9                                                 Total Cells × 10.sup.3 /mg:                                                           702.8                                                                              717.6 618.9                                                (% of Normal Control Value):                                                                (100%)                                                                             (102%)                                                                              (88%)                                                Monocytes: %: 2.4  3.0   2.5                                                  Total Cells × 10.sup.3 /mg:                                                           58.5 41.4  35.3                                                 (% of Normal Control Value):                                                                (100%)                                                                             (71%) (60%)                                                Megakaryocytes: %:                                                                          0.4  0.2   0.3                                                  Total Cells × 10.sup.3 /mg:                                                           9.8  2.9   4.2                                                  (% of Normal Control Value):                                                                (100%)                                                                             (29%) (43%)                                                Erythroblasts:                                                                %:            1.9  2.3   2.1                                                  Total Cells × 10.sup.3 /mg:                                                           46.4 31.7  29.6                                                 (% of Normal Control Value):                                                                (100%)                                                                             (68%) (64%)                                                Erythroid cells: %:                                                                         9.4  9.9   9.7                                                  Total Cells × 10.sup.3 /mg:                                                           229.4                                                                              136.6 136.0                                                (% of Normal Control Value):                                                                (100%)                                                                             (60%) (59%)                                                __________________________________________________________________________

The results presented in TABLE 71 show that the 5-FU dose schedulereduced the total number of all precursor cell types in the bone marrowexcept myeloblasts and promyeloblasts, and the results presented inTABLE 72 show that peripheral blood leukocytes were reduced to 44% ofnormal. In the bone marrow (TABLE 71), treatments with L--Glu--L--Trpresulted in: i) an increase in the numbers of myelocytes/neutrophils,stab and segmented neutrophils, and megakaryocytes; ii) no markedchanges in the total numbers of myelokaryocytes, reticular cells,monocytes, erythroblasts, and erythroid cells; and, iii) a decrease inthe absolute numbers of myeloblasts, promyeloblasts, eosinophils, andlymphocytes. In the peripheral blood (TABLE 72) the followingobservations were recorded: i) the total number of neutrophils increased(i.e., in parallel with the observed increase in bone marrowneutrophils); ii) the absolute number of monocytes dropped slightly, butinsignificantly, since the percentage of monocytes in peripheral bloodwas about 2-3% before and after treatment; and, iii) the total number ofperipheral blood lymphocytes increased (i.e., in parallel with theobserved decrease in bone marrow lymphocytes). The observed decrease inbone marrow lymphocytes and increase in peripheral blood lymphocytes wasconsistent with L--Glu--L--Trp mobilization of these cells into theperipheral blood. The effects of the L--Glu--L--Trp treatment on otherperipheral blood leukocyte populations in the 5-FU immunosuppressed miceare summarized in TABLE 72.

                                      TABLE 72                                    __________________________________________________________________________    Peripheral Blood Leukocyte Populations in 5-FU Suppressed                     CBA Mice Treated with L--Glu--L--Trp                                                       Normal                                                                             Negative                                                                            Experimental                                                       Control                                                                            Control                                                                             (5-FU + L--Glu--L--Trp                                Cell Type    (untreated)                                                                        (5-FU only)                                                                         Therapy)                                              __________________________________________________________________________    Peripheral Blood Leukocytes:                                                  Total Cells × 10.sup.9 /L:                                                           5.72 2.51  2.98                                                  (% of Normal Control Value):                                                               (100%)                                                                             (44%) (52%)                                                 Lymphocytes: %:                                                                            47.3 83.9  41.9                                                  Total Cells × 10.sup.9 /L:                                                           2.71 2.10  2.52                                                  (% of Normal Control Value):                                                               (100%)                                                                             (78%) (93%)                                                 Stab. Neutrophils: %:                                                                      16.1 3.7   7.9                                                   Total Cells × 10.sup.9 /L:                                                           0.92 0.09  0.24                                                  (% of Normal Control Value):                                                               (100%)                                                                             (10%) (26%)                                                 Segmented Neutrophils: %:                                                                  33.9 3.3   14.6                                                  Total Cells × 10.sup.9 /L:                                                           1.93 0.08  0.43                                                  (% of Normal Control Value):                                                               (100%)                                                                             (4%)  (22%)                                                 Monocytes:   1.86 3.35  2.5                                                   %:           0.11 0.08  0.02                                                  Total Cells × 10.sup.9 /L:                                                           (100%)                                                                             (73%) (18%)                                                 (% of Normal Control Value):                                                  Eosinophils: 0.2  0.2   0.3                                                   %:           0.01 0.01  0.01                                                  Total Cells × 10.sup.9 /L:                                                           (100%)                                                                             (100%)                                                                              (100%)                                                (% of Normal Control Value):                                                  Blast Cells: 0    5.2   0.3                                                   %:           0    0.13  0.01                                                  Total Cells × 10.sup.9 /L:                                              __________________________________________________________________________

It is noteworthy that treatments with L--Glu--L--Trp were observed toincrease the numbers of bone marrow megakaryocytes and neutrophils andalso peripheral blood neutrophils, since these changes may provide animmunosuppressed subject an increased number of peripheral bloodplatelets and an increased measure of innate resistance to infection.

EXAMPLE 36 Effects of Synthetic Peptide L--Glu--L--Trp on Immune Statusin Immunocompromised Rats

An immunocompromised state was established over a period of 80 days inexperimental rats by administration of cortisone acetate at a dosage of25 mg/kg twice weekly with tetracycline hydrocholoridepo in drinkingwater. In the experimental group, L--Glu--L--Trp was administered inthree treatment courses, each course consisting of 10 daily iminjections of 10 μg/kg or 100 μg/kg, and each course of treatmentseparated from the next course by a 1 month interval. In the controlgroup, saline was administered in the same three treatment courseschedule. Differential blood counts were conducted on day 0 (NormalControl), day 59, and day 79 of the study. L--Glu--L--Trp treatment atdosages of both 10 μg/kg and 100 μg/kg significantly (p<0.05) increasedthe number of leukocytes, monocytes, and lymphocytes in the peripheralblood of cortisone-suppressed rats relative to the saline treatedcontrols. Results obtained with the 10 μg/kg treatment dose (presentedin TABLE 73) were not markedly different than those obtained with the100 μg/kg dose.

                                      TABLE 73                                    __________________________________________________________________________    Effects of L--Glu--L--Trp Treatments on Peripheral Blood                      Leukocytes in Immunocompromised Cortisone Suppressed Rats.                                 Saline                                                                              Saline      L--Glu--L--Trp                                              Control                                                                             Control     Treatment (10 μg/kg)                        Cell Type    Day 0 Day 59                                                                              Day 79                                                                              Day 59                                                                              Day 79                                   __________________________________________________________________________    Leukocytes:                                                                   Total Cells × 10.sup.9 /L:                                                           16.5 ± 1.3                                                                       10.5 ± 0.6*                                                                      5.4 ± 0.6*                                                                       12.4 ± 1.8                                                                       8.06 ± 0.9*†                   (% of Normal Control Value):                                                               (100%)                                                                              (64%) (33%) (75%) (49%)                                    Stab Neutrophils: %:                                                                       3.9 ± 0.9                                                                        6.9 ± 1.3                                                                        7.6 ± 1                                                                          4.7 ± 0.6                                                                        4.4 ± 0.7†                     Total Cells × 10.sup.9 /L:                                                           0.6 ± 0.2                                                                        0.7 ± 0.1                                                                        0.4 ± 0.1                                                                        0.6 ± 0.1                                                                        0.35 ± 0.1                            (% of Normal Control Value):                                                               (100%)                                                                              (117%)                                                                              (67%) (100%)                                                                              (58%)                                    Segmented Neutrophils: %:                                                                  27 ± 0.6                                                                         57.7 ± 2*                                                                        56 ± 2*                                                                          26.7 ± 1†                                                                 24.6 ± 1.3†                    Total Cells × 10.sup.9 /L:                                                           4.5 ± 0.4                                                                        6 ± 0.4                                                                          3 ± 0.3*                                                                         3.3 ± 0.4†                                                                2 ± 0.2*†                      (% of Normal Control Value):                                                               (100%)                                                                              (133%)                                                                              (67%) (73%) (44%)                                    Eosinophils: %:                                                                            2.6 ± 0.5                                                                        2.4 ± 0.6                                                                        6.4 ± 0.8*                                                                       4.3 ± 0.4†                                                                4 ± 0.9                               Total Cells × 10.sup.9 /L:                                                           0.4 ± 0.1                                                                        0.3 ± 0.1                                                                        0.3 ± 0.1                                                                        0.6 ± 0.1                                                                        0.3 ± 0.1                             (% of Normal Control Value):                                                               (100%)                                                                              (75%) (75%) (150%)                                                                              (75%)                                    Basophils: %:                                                                              0.4 ± 0.2                                                                        0.7 ± 0.2                                                                        0.3 ± 0.2                                                                        0.4 ± 0.2                                                                        0.3 ± 0.2                             Total Cells × 10.sup.3 /mg:                                                          0.07 ± 0.04                                                                      0.07 ± 0.02                                                                      0.02 ± 0.01                                                                      0.05 ± 0.03                                                                      0.02 ± 0.02                           (% of Normal Control Value):                                                               (100%)                                                                              (100%)                                                                              (29%) (71%) (29%)                                    Monocytes: %:                                                                              4.7 ± 0.6                                                                        3.7 ± 0.6                                                                        8.7 ± 1.3*                                                                       7 ± 0.9*†                                                                 8 ± 0.8*                              Total Cells × 10.sup.9 /L:                                                           0.7 ± 0.1                                                                        0.4 ± 0.1*                                                                       0.5 ± 0.1*                                                                       0.92 ± 0.2†                                                               0.7 ± 0.1                             (% of Normal Control Value):                                                               (100%)                                                                              (57%) (71%) (128%)                                                                              (100%)                                   Lymphocytes: %:                                                                            61 ± 1.3                                                                         29 ± 2*                                                                          21 ± 2*                                                                          57 ± 1.5†                                                                 59 ± 2†                        Total Cells × 10.sup.9 /L:                                                           10 ± 0.8                                                                         3 ± 0.3*                                                                         1.1 ± 0.1*                                                                       7 ± 1†                                                                    4.7 ± 1†                       (% of Normal Control Value):                                                               (100%)                                                                              (30%) (2%)  (11%) (7.7%)                                   __________________________________________________________________________     *, statistically significant difference (p < 0.05) from the values            recorded on day 0;                                                            †statistically significant difference (p < 0.05) from the values       recorded in saline control group on day 59; day 0 values for animals in       the L--Glu--L--Trp treated group were not statistically different from        those in the saline treated group.                                       

EXAMPLE 37 Stimulation of Natural Killer Cell Cytotoxic Activity byL--Glu--L--Trp Treatments

Natural killer cells (i.e., NK lymphocytes) are considered by many to beone element of innate resistance to infection. L--Glu--L--Trp was testedunder GLP conditions for its ability to effect a change in the activityof splenic natural killer (NK) lymphocytes. L--Glu--L--Trp was injecteddaily on each of 7 consecutive days ip into C3H/HeJ mice at a dose of 1,10, or 1000 ,μg/kg (0.5 mL/mouse). Cyclophosphamide (50 μg/kg), orsaline, were used as reference treatments. On day 8, spleen cells wereprepared and diluted to 5×10⁶ cells/ml. YAC-1 target cells weresuspended at 5×10⁶ cells/mL in Tris buffer containing 200 μCi/ml of ⁵¹Cr. After a 1 hour incubation at 37° C. the YAC-1 cells (target cells)were washed 3 times, and resuspended at a concentration of 1×10⁵cells/mL. Each 100 μL aliquot of target cells was mixed with a 100 μLaliquot of splenocytes (effector cells) in a well of a 96 wellmicrotiter plate. Spontaneous release from the target cells wasdetermined by incubating without effector cells; maximal release wasdetermined by lysing target cells with 1N HCl. Cytotoxic killing oftarget cells was determined after 4 hours incubation at 37° C. Specific⁵¹ Cr-release was calculated according to the following formula:##EQU1##

The mean value for each group of 10 animals. The results are presentedin TABLE 74.

                  TABLE 74                                                        ______________________________________                                        NK Activity of Splenocytes from L--Glu--L--Trp Treated Mice.                                    Cytotoxicity                                                                            % Cytotoxicity                                                                          Change                                  Group  Treatment    Dose    (mean +/- S.D.)                                                                         (%)*                                    ______________________________________                                        1      Saline        0       8.5 ± 2.0                                                                           --                                      2      Glu--Trp      1      10.7 ± 1.4                                                                           25 ↑                              3                   10      14.3 ± 1.7                                                                           67 ↑                              4                   1000    14.9 ± 2.9                                                                            75* ↑                            5      Cyclophosphamide                                                                           50       6.4 ± 0.7                                                                           -25 ↓                            ______________________________________                                         *statistically significant compared to saline control; p < 0.05          

The results presented in TABLE 74 show that 7 days treatment withL--Glu--L--Trp increased the apparent NK cytotoxic activity of murinesplenocytes by 25 to 75%. As expected, cyclophosphamide treatmentdecreased NK activity.

EXAMPLE 38 Stimulation of Anti-Viral Activity by Treatments withL--Glu--L--Trp

Innate mechanisms of anti-viral immunity are important to survival ofthe host. The Rauscher murine leukemia virus (MuLV) is an example of ahighly virulent animal retrovirus infection, first isolated andcharacterized by Dr. Rauscher from BALB/c mouse tissues in 1962.Although anti-viral agents may exhibit viricidal activity in this model,many (if not most) immunomodulatory compounds lack significant activityin this animal model. The virus employed in present GLP/blinded studywas a murine leukemia complex known to induce progressiveerythroleukemia in mice (i.e., Rauscher MULV and replication-defectiveRauscher spleen focus-forming virus). Disease manifested within about 6days with death by about 50 days. Measurements of splenomegaly, bloodreverse transcriptase, virus plaque assays (i.e., XC-plaque assay forviremia using SC-1 host cells) and serum anti-viral antibody titers wereused to assess infection. Spleens achieved about 2.0 grams in weightwithin 21 days of infection, as compared with a normal spleen weights inan uninfected animals of about 0.1 gram.

Mice were inoculated with Rauscher MuLV complex (0.1 ml/7.8×10⁴ PFU/mL)on day 0 and sacrificed on day 21. Test groups were comprised of 10 miceand control groups 6 mice. Control articles included saline and AZT (indrinking water at about 1.25 mg/mouse/day≈63 mg/kg/day/mouse).L--Glu--L--Trp was administered ip on each of 5 consecutive days todifferent groups of animals at doses of either 1, 10, 50, 100 or 500μg/kg. A non-infected group of mice was also sacrificed on day 21 toprovide normal control values.

                                      TABLE 75                                    __________________________________________________________________________    Stimulation of an Anti-Viral Response to Rauscher MuLV in Mice Treated        with L--Glu--L--Trp                                                                            Anti-Viral Activity                                                       Dose                                                                              Spleen Weight                                                                        Reduction                                                                              Viremia                                      Group  Treatment*                                                                          (mg/kg)                                                                           (mean ± SD)*                                                                      Splenomegaly (%)                                                                       (log.sub.10 PFU/ml ± SEM)                 __________________________________________________________________________     1     None  0   0.13 ± 0.02                                                                       --       --                                           (non-infected)                                                                 2     None  0   0.35 ± 0.1                                                                        --       --                                            3     Saline                                                                              0    0.4 ± 0.15                                                                       --       --                                            4     AZT   200 0.31 ± 0.09                                                                       46       ND                                            5           100 0.29 ± 0.02                                                                       52       ND                                            6           50  0.35 ± 0.17                                                                       33       ND                                            7           25  0.31 ± 0.05                                                                       46       ND                                            8     Poly(IC)                                                                            20  0.37 ± 0.22                                                                       27       ND                                            9     Saline                                                                              0   0.62 ± 0.36                                                                       --        4.06 ± 0.369.sup.                        10     Poly(IC)                                                                            20  0.72 ± 0.29                                                                       -78      ND                                           11     Glu--Trp                                                                            5   0.44 ± 0.15                                                                        6        3.43 ± 0.368.sup.†                12           1.75                                                                              0.45 ± 0.39                                                                        3       ND                                           13           0.5 0.48 ± 0.3                                                                        -12      3.31 ± 0.39.sup.†                  14           0.17                                                                              0.42 ± 0.26                                                                       12       ND                                           15           0.05                                                                              0.36 ± 0.11                                                                       30        2.99 ± 0.28.sup.† **              16           0.02                                                                              0.42 ± 0.32                                                                       12       ND                                           17           0.005                                                                             0.47 ± 0.29                                                                       -3       3.25 ± 0.27.sup.†                  18           0.002                                                                             0.49 ± 0.31                                                                       -9       ND                                           __________________________________________________________________________     *Groups 4-8 were treated with AZT or poly(IC) on days 0, 1, 2, 3, and 4;      Groups 10-18 were treated on days -2, -1, 0, +1, and +2 with the indicate     compounds;                                                                    **1 log reduction in virus titer is about 99% virus kill;                     .sup.† Probability MannWhitney U Test as follows: namely, group 11     (P.sup.3 = 0.315); group 13 (P.sup.3 = 0.315); group 15 (P.sup.3 = 0.036)     group 16 (P.sup.3 = 0.143).                                              

The results presented in TABLE 75 show that treatment withL--Glu--L--Trp significantly decreased viremia (i.e., at a dosage of 50μg/kg), even in animals exhibiting splenomegaly.

EXAMPLE 39 Stimulation of Changes in Cyclic Nucleotides (cAMP/cGMP)Following Treatments with L--Glu--L--Trp

Normal guinea pigs (10/group) were treated with 10 μg/kg L--Glu--L--Trpdaily on each of five consecutive days and then spleens were removed,lymphocytes isolated, and cyclic AMP and GMP levels determined. Toevaluate the effects of L--Glu--L--Trp on immune cells in the face of anongoing anaphylactic response, splenic lymphocytes were isolated fromguinea pigs sensitized for anaphylaxis, or following induction ofanaphylactic shock.

                                      TABLE 76                                    __________________________________________________________________________    L--Glu--L--Trp Induced Increases in Splenic cAMP and cGMP                                       Dose                                                                              Cyclic Nucleotide Content (pmole/10.sup.7 cells)        Group                                                                             Sensitized                                                                         Anaphylaxis                                                                         EW*                                                                              (μg/kg)                                                                        cAMP  cGMP  cAMP/cGMP                                   __________________________________________________________________________    1   -    -     -  0   56 ± 6                                                                           1.8 ± 0.2                                                                        31 ± 3                                   2   +    -     -  0   157 ± 13*                                                                        5.7 ± 0.3*                                                                       27 ± 3                                   3   +    -     +  10   250 ± 30**                                                                      11.2 ± 0.9**                                                                     22 ± 2                                   4   +    +     -  0   117 ± 10*                                                                        4.2 ± 0.3*                                                                       28 ± 2                                   5   +    +     +  10   210 ± 30**                                                                       7.9 ± 0.5**                                                                     26 ± 3                                   __________________________________________________________________________     *p < 0.05 compared with Group #1;                                             **p < 0.05 compared with Group #2;                                            .sup.† p < 0.05 compared with Group #4                            

The results presented in TABLE 76 show, on a macroscopic level, that:(a) the intensive immunization scheme required to induce sensitizationfor anaphylaxis induced a significant increase in the total levels ofcAMP and cGMP in splenic lymphocytes, i.e., control animals in Group 2;and, (b) induction of anaphylaxis (Group 4) significantly decreased thelevels of cAMP from the levels in the sensitized animals (Group 2).Treatments with L--Glu--L--Trp significantly elevated the levels of cAMPand cGMP in sensitized animals (Group 3) as well as anaphylactic animals(Group 5). The results suggest a significant stimulatory effect ofL--Glu--L--Trp on lymphocytes in anaphylactic animals. Anaphylaxis isknown to be accompanied by release of negative regulators of lymphocyteand macrophage function. Evidence of a stimulatory effect ofL--Glu--L--Trp treatments in this model is presently considered highlysuggestive of a possible therapeutic efficacy in a variety of acute andchronic disease settings where lymphocyte and monocyte function is knownto be down-regulated, e.g., septic shock, acute respiratory distresssyndrome, asthma, and tuberculosis infection. Direct testing of efficacyin a tuberculosis animal model follows in Example 40, below.

EXAMPLE 40 Treatment of Murine Mycobacterium bovis Infection withL--Glu--L--Trp

The effects of L--Glu--L--Trp treatments on anti-bacterial cellularimmunity were evaluated using a murine model of mycobacterial infection.Mice were inoculated iv with 0.1 mg (dry weight) of a mouse-passaged andadapted virulent laboratory strain of M. bovis. L--Glu--L--Trp wasadministered daily and the effects compared with those achieved withisoniazid (INH; also administered daily). Doses of L--Glu--L--Trp were1, 10 and 100 μg/kg ip and INH was administered at 5 mg/kg sc. Survivalwas assessed out to 62 days post-infection. The treatment scheduleincluded prophylactic and therapeutic regimens as follows: (i)prophylactic/therapeutic treatments with L--Glu--L--Trp consisted ofadministering the test dose ip daily on each of 5 consecutive days ofeach week of the study starting 3 days before infecting the animals;and, (ii) therapeutic treatments with L--Glu--L--Trp consisted ofadministering the test dose ip daily on each of 5 consecutive days ofeach week starting at week 3 of the study. The survival statistics at 27days are presented in TABLE 77.

                                      TABLE 77                                    __________________________________________________________________________    Survival of Mice after Infection with a Lethal Dose                           of M. bovis and L--Glu--L--Trp Therapy                                                         Dose         Survival                                        Group                                                                             Isoniazid                                                                          Therapy.sup.a                                                                      EW.sup.b                                                                         (μg/kg)                                                                        No./grp.sup.c                                                                      Mean                                                                              Median                                                                             p value.sup.d                              __________________________________________________________________________    1   -    None -  0   43   27 ± 1                                                                         26 ± 1                                                                          -                                          2   -    PDR  +  100 32   31 ± 1                                                                         28 ± 1                                                                          ns                                         3   -    PDR  +  10  32   28 ± 1                                                                         25 ± 1                                                                          ns                                         4   -    PDR  +  1   33   39 ± 2                                                                          46 ± 10                                                                        <0.05                                      5   -    TDR  +  100 30   36 ± 3                                                                         28 ± 3                                                                          <0.05                                      6   -    TDR  +  10  30   30 ± 2                                                                         26 ± 1                                                                          ns                                         7   -    TDR  +  1   30   33 ± 1                                                                         34 ± 3                                                                          <0.001                                     8   +    None -  0   25   57 ± 2                                                                         -    <0.001                                     __________________________________________________________________________     .sup.a PDR, prophylactic treatment regimen; TDR, therapeutic treatment        regimen;                                                                      .sup.b EW = L--Glu--L--Trp;                                                   .sup.c No./Tot. = number of survivors/total evaluable animals;                .sup.d X.sup.2 = Chi square significance for Groups 2-8 as compared with      Control Group 1.                                                         

The results in TABLE 77 show that prophylactic treatment with only theL--Glu--L--Trp dipeptide at the lowest dose tested, i.e., 1 μg/kg (Group4), significantly increased survival, and therapeutic treatment at 1μg/kg (Groups 5 and 7) also significantly increased survival. Theresults are particularly noteworthy because they compare the effects ofa proven anti-microbial compound (i.e., isoniazid) with those of animmune modulator (i.e., L--Glu--L--Trp).

EXAMPLE 41 Treatment of Murine Candida albicans Infection withL--Glu--L--Trp

In this animal model, inoculated C. albicans cells germinate rapidlywithin 2-4 hours as evidenced by formation of germ tubes. Administrationof anti-mycotic preparations (e.g., amphotericin) or certainimmunomodulators can entirely block germ tube development. Germ tubedevelopment was monitored histologically and mean survival time servedas the endpoint in Study 41A and mean survival time in Study 41B.

Study 41A: Mice were inoculated ip with 5×10⁶ C. albicans cells that hadbeen germinated rapidly in vitro by shifting from Sabouraud agar (tubes)to sterile saline and rotary culture in order to favor development ofgerm tubes (i.e., lateral pseudohyphal filaments protruding from thespherical yeast cells). Six hours after inoculation mice were euthanizedthe epiploons removed and fixed in 10% formalin for histologicalanalysis, using periodic acid Schiff's to visualize the yeast in thetissues. The percentage of germ-tube positive yeast cells was determinedmicroscopically at 400× in the histological preparations.

Five different doses of L--Glu--L--Trp were tested by ip administration:i.e., 300, 100, 10, 1, and 0.1 μg/kg. Amphotericin was used as apositive control and saline as a negative control. The treatments wereadministered daily on each of the three consecutive days with the lastip injection being 3 hours before inoculating the yeast. At least 300yeast cells (about 10 microscopic fields) were evaluated. The totalnumber of fungal cell, number of non-germinated forms, and number ofcells were germ tubes were all determined. Results are expressed as themean percentage of yeast cells with germ tubes±SEM for each group ofanimals. A value (Student's t-test) of p<0.05 was consideredstatistically significance.

                  TABLE 78                                                        ______________________________________                                        Survival of Mice after Infection with a Lethal Dose                           of C. albicans and L--Glu--L--Trp Therapy                                                                     Dose   Germ                                   Group  Amphotericin                                                                             Therapy.sup.a                                                                          EW.sup.b                                                                           (/kg)  Tube (%)                               ______________________________________                                        1      -          None     -    0        37                                   2B     +          Control  -    0.063                                                                              mg  20*                                  2C     +                   -    0.125                                                                              mg  17*                                  2D     +                   -    0.25 mg  14*                                  2E     +                   -    0.5  mg   3*                                  2F     +                   -    1    mg   1*                                  3A     -          PDR      +    0.1  μg                                                                             35*                                  3B     -                   +    1    μg                                                                             34*                                  3C     -                   +    10   μg                                                                             35*                                  3D     -                   +    100  μg                                                                             26*                                  3E     -                   +    300  μg                                                                             37                                   ______________________________________                                         *p < 0.05 compared to saline control group #1;                                .sup.a PDR, phophylactic drug regimen;                                        .sup.b EW = L--Glu--L--Trp                                               

Study 41B: Using a standardized inoculum (i.e., 10⁸ cells/mL), the LD₉₅value (=14.9×10⁶ cells/animal) was determined by injecting differentnumbers of C. albicans cells iv into mice and recording deaths at 20days. L--Glu--L--Trp was administered ip at doses of 1, 10, 100 and 300μg/kg in either a prophylactic or therapeutic drug regimen (abbreviatedPDR or TDR, respectively). Prophylaxis consisted of 3 ip injections on 3consecutive days, with the last injection being 3 hours before injectingthe C. albicans cells iv. Therapy consisted of 10 consecutive daily ipinjections starting 24 hours after the iv injection of the C. albicanscells. Mean survival was determined at 10 days, and as an independentmonitor for infection, kidneys were removed from nine parallel groups(12 animals each) of infected animals at 5 days and aftermicrobiological isolation the numbers of C. albicans cultures counted.The results are summarized in TABLE 79.

                                      TABLE 79                                    __________________________________________________________________________    Effects of L--Glu--L--Trp on Mean Survival Time in Mice with Disseminated     Candidiasis                                                                                      Dose                                                                              Survival (days)d      Kidney                           Group                                                                             Amphotericin                                                                         Therapy.sup.a                                                                      EW.sup.b                                                                         (μg/kg)                                                                        No./grp.sup.c                                                                      Mean % 6d                                                                             X.sup.2                                                                          % 8d                                                                             X.sup.2                                                                          Infection.sup.e                  __________________________________________________________________________    1   -      None -   0  20   5.4 ± 1.6                                                                       30 -   0 -  100%                             2   -      PDR  +   1  10   6.2 ± 2.0                                                                       50 ns 20 <0.05                                                                            58%                              3   -      PDR  +   10 10   6.4 ± 2.2                                                                       60 ns 20 <0.05                                                                            58%                              4   -      PDR  +  100 10   7.9 ± 1.9                                                                       80 <0.05                                                                            40 <0.05                                                                            75%                              5   -      PDR  +  300 10   7.8 ± 2.1                                                                       80 <0.05                                                                            50 <0.05                                                                            58%                              6   -      TDR  +   1  10   7.3 ± 2.7                                                                       60 ns 50 <0.05                                                                            67%                              7   -      TDR  +   10 10   6.2 ± 2.2                                                                       50 ns 30 <0.05                                                                            50%                              8   -      TDR  +  100 10   7.8 ± 1.9                                                                       80 <0.05                                                                            50 <0.05                                                                            75%                              9          TDR  +  300 10   7.8 ± 2.1                                                                       80 <0.05                                                                            40 <0.05                                                                            67%                              __________________________________________________________________________     .sup.a PDR, phophylactic treatment regimen; TDR, therapeutic treatment        regimen;                                                                      .sup.b EW = L--Glu--L--Trp;                                                   .sup.c No./Tot. = number of survivors/total evaluable animals;                .sup.d X.sup.2 = Chi square significance for Groups 2-8 as compared with      Control Group 1;                                                              .sup.e kidney infection = greater than 8 logs of C. albicans cultured fro     kidney samples removed from mice in a parallel group of 12 animals on day     5.                                                                       

The results presented in TABLE 79 show that prophylactic and therapeutictreatments with L--Glu--L--Trp significantly increased the number ofanimals surviving at days 6 and 8, and reduced the incidence of kidneyinfection at day 5.

EXAMPLE 42 Treatment of Rat Pneumocystis carini Infection withL--Glu--L--Trp

Corticosteroids were administered to rats to induce an immunocompromisedstate in which an opportunistic infection with Pneumocystis carini couldbe established. The immunocompromised state was induced by administeringcortisone acetate im twice weekly at a dose of 25 mg/kg over the entire12 week course of the study. Pneumocystis carini lung infection wasinitiated by intranasal instillation on day 0. Prophylactic tetracyclinewas administered po in the drinking water (500 mg/mL), and peripheralblood differential counts were monitored over the next 12 weeks. Incontrol (untreated) Pneumocystis-infected animals, differential counts,body weight, and lymphocyte counts began dropping at about 30-39 daysand lung infection was evident histologically by about day 60-69. At day80, tetracycline treatment was discontinued in all groups. By day 80 inthis animal model, mean body weight of the animals had dropped byapproximately 30% from the values recorded on day 0. Groups werecomprised of 15 rats each.

L--Glu--L--Trp treatments were administered according to the followingprotocol: 10 consecutive daily im injections of 10 μg/kg or 100 μg/kg ofL--Glu--L--Trp on days 0 through 9, 30 through 39, and 60 through 69 ofthe study. Differential cell counts in peripheral blood (TABLE 80) andlung histology (i.e., with enumeration of the number of P. carinii/10microscopic fields--at 400× magnification) were used to monitor thecourse of the opportunistic infection.

                                      TABLE 80                                    __________________________________________________________________________    Differential Cell Counts on Peripheral blood obtained from Mice at 79         Days after Lung                                                               Infection with a Lethal Dose of Pneumocystis carinii and Treatments with      L--Glu--L--Trp.                                                                      Dose                                                                              Total Leukocytes                                                                         Lymphocytes                                                                              Neutrophils                                                                              Lung                              Group                                                                             EW.sup.a                                                                         (μg/kg)                                                                        (× 10.sup.6 /ml)                                                             (% normal).sup.b                                                                    (× 10.sup.6 /ml)                                                             (% normal).sup.b                                                                    (× 10.sup.6 /ml)                                                             (% normal).sup.b                                                                    (bacteria).sup.c                  __________________________________________________________________________    1   -   0  5.4 ± 1.5                                                                       31    1.1 ± 0.4                                                                       11    3.0 ± 0.9                                                                       62    160 ± 80                       2   +   10 8.1 ± 2.3                                                                       47    4.7 ± 1.2                                                                       45    2.0 ± 0.7                                                                       41    53 ± 32                        3   +  100 7.8 ± 1.8                                                                       45    4.7 ± 1.4                                                                       45    1.5 ± 0.5                                                                       31    70 ± 33                        __________________________________________________________________________     .sup.a EW = L--Glu--L--Trp treatment;                                         .sup.b % normal = % of normal leukocyte values recorded on day 0;             .sup.c Lung bacteria = mean ± S.E. number of P. carinii bacteria per 1     sections (i.e., at a magnificant of 400X).                               

The results presented in TABLE 80 show that long-term cortisoneadministration induced the desired reduction in peripheral bloodleukocytes (i.e., 31% of normal), lymphocytes (i.e., 47% of normal) andneutrophils (i.e., 62% of normal), and resulted in establishment of anopportunistic lung infection. Treatments with L--Glu--L--Trp helpedmaintain leukocyte (i.e., 45-47% of normal) and lymphocyte counts (i.e.,45% of normal), but not systemic neutrophil counts. Numbers of lungbacteria were reduced by L--Glu--L--Trp treatments at the 10 μg/kg and100 μg/kg doses (i.e., to about 33% and 43%, respectively, of the salinetreated control Group 1).

EXAMPLE 43 Stimulation of Anti-Tumor Immunity by Treatment withL--Glu--L--Trp

Anti-tumor activity of L--Glu--L--Trp was evaluated using the murineSarcoma 180 tumor (ATCC CCL-8 CCRF S-180 II) injected at 2×10⁶ cells/0.1mL im into each rear flank of Swiss-Webster mice. Groups consisted of 10animals. L--Glu--L--Trp was administered in a single 0.1 mL dose ofeither 10 μg/kg, 75 μg/kg, 250 μg/kg or 1000 μg/kg. Tumor size wasevaluated by surgically removing and weighing the affected limbs, andcomparing the weight with the weight of normal control (non-tumor)limbs. The first prophylactic drug regimen (PDR-1) consisted of 5consecutive daily ip injections commencing on day -5 and ending of day-1. The second prophylactic drug regimen (PDR-2) consisted of 5consecutive daily im injections to the left rear flank (tumor site)beginning on day -5 and ending on day -1. Sarcoma 180 cells wereinjected im on day 0. Saline 0.1 mL served as the negative control.

                                      TABLE 81                                    __________________________________________________________________________    Effect of L--Glu--L--Trp Treatments on Sarcoma 180 Bilateral Tumor Size       Treatment Dose                                                                              Leg Weight (g)                                                                          Mean Tumor Weight.sup.a                                                                 Percent of Control                          Group                                                                              Regimen                                                                            (μg/kg)                                                                        Left Right                                                                              Left Right                                                                              Tumor Weight.sup.b                          __________________________________________________________________________    1A   None  0  1.2 ± 0.1                                                                       1.2 ± 0.2                                                                       0    0    --   --                                     (normal)                                                                      1B   None  0  3.7 ± 0.6                                                                       3.5 ± 0.7                                                                       2.5  2.3   0    0                                     (tumor)                                                                       2    PDR-1                                                                              10  4.2 ± 0.9                                                                       4.1 ± 0.7                                                                       3.0  2.9  120  126                                    3    PDR-1                                                                              75  4.2 ± 0.8                                                                       4.2 ± 0.8                                                                       3.0  3.0  120  130                                    4    PDR-1                                                                              250 3.5 ± 1.0                                                                       3.1 ± 0.6                                                                       2.3  1.9  92    83                                    5    PDR-1                                                                              1000                                                                              2.5 ± 0.7                                                                       2.2 ± 0.5                                                                       1.3  1.0  52    43                                    6    PDR-2                                                                              10  3.6 ± 0.7                                                                       3.8 ± 0.7                                                                       2.4  2.6  96   113                                    7    PDR-2                                                                              75  3.6 ± 0.7                                                                       3.5 ± 0.3                                                                       2.4  3.3  96   143                                    8    PDR-2                                                                              250 3.0 ± 0.1                                                                       2.3 ± 0.5                                                                       1.8  1.1  72    48                                    9    PDR-2                                                                              1000                                                                              2.4 ± 0.5                                                                       2.5 ± 0.5                                                                       1.2  1.3  48    57                                    __________________________________________________________________________     .sup.a Mean Tumor weight = (mean leg weight treated - mean leg weight         normal control);                                                              .sup.b Inhibition = (tumor weight treated/tumor weight control) ×       100%                                                                     

The results presented in TABLE 81 show that prophylactic treatments withL--Glu--L--Trp ip or im at doses of 250 μg/kg and 1000 μg/kg inhibitedsubsequent in tumor growth. Interestingly, it appeared possible toinvoke systemic inhibitor effects from treatments delivered at a localim site, because the im treatments delivered into the left flankinhibited subsequent tumor growth in the right flank (i.e., groups 8 and9). The results are consistent with stimulation of systemiccell-mediated tumor immune mechanisms.

EXAMPLE 44 Stimulation of DTH Sensitization by Treatment withL--Glu--L--Trp

Effects of L--Glu--L--Trp treatments on cell mediated immunity wereevaluated by testing its effects on induction of a delayed-typehypersensitivity response in two different studies involvingsensitization of mice to oxazolone and guinea pigs to tuberculin.

In the first study L--Glu--L--Trp was administered ip at a dose of 10μg/kg, or 1000 μg/kg one hour before a sensitizing dose of oxazolone(0.1 mL of a 5% solution) was applied to the abdominal surface of amouse. Seven days later skin sensitivity was measured by administering25 μL of a 2% oxazolone solution to the right mouse ear and a sample ofthe diluent solution, as a control, to the left ear. Ear thickness wasmeasured at 24 hours using a Dyer Model micrometer. Three groups of 10animals each were tested and the results are summarized in TABLE 82.

                  TABLE 82                                                        ______________________________________                                        L--Glu--L--Trp Induced DTH Sensitivity of Oxazolone in Mice                                                Dose  Mean Ear Thickness ±                    Group Sensitized                                                                             Oxazolone                                                                              EW*  (μg/kg)                                                                          S.D. (μm)                               ______________________________________                                        1     -        +        -      0   17 ± 2                                  2     +        +        +     10   17 ± 1                                  3     +        +        +    1000  24 ± 1                                  ______________________________________                                    

The results presented in TABLE 82 show an increase in DTH sensitivity tooxazolone in the group of mice treated with the 1000 μg/ML dose ofL--Glu--L--Trp one hour prior to administering antigen.

In the second study, affects of L--Glu--L--Trp on DTH sensitivity wereinvestigated in guinea pigs sensitized to tuberculin. L--Glu--L--Trp wasadministered ip at 10 μg/kg or 1000 μg/kg on the day of an intradermalsensitization with tuberculin antigen. DTH skin sensitivity was measuredon day 10 and day 20, and the results are summarized in TABLE 83.

                                      TABLE 83                                    __________________________________________________________________________    L--Glu--L--Trp Induced DTH Sensitivity of Oxazolone in Mice                   tuberculin                                                                              skin test                                                                             Dose                                                                              Mean Skin Test Diameter ± S.D. (mm)                  Group                                                                              Sensitized                                                                         tuberculin                                                                         EW*                                                                              (μg/kg)                                                                        Day 10  Day 20                                          __________________________________________________________________________    1    +    +    -    0 8.7 ± 0.7                                                                          7.4 ± 0.7                                    2    +    +    +   10 14.4 ± 1.3*                                                                        15.1 ± 1.4*                                  3    +    +    +  1000                                                                              15.2 ± 1.3*                                                                        13.9 ± 1.1*                                  __________________________________________________________________________     *p < 0.05 compared with Group #1                                         

The results presented in TABLE 83 show that L--Glu--L--Trp induced astatistically significant increase in the DTH response measured totuberculin antigen as measured on day 10 or day 20 after immunization,and as compared with control tuberculin-sensitized animals.

EXAMPLE 45 Effects of L--Glu--L--Trp on Survival in a Model of LPS Shock

The LD₉₉ and LD₅₀ values for LPS-induced endotoxic shock (a.k.a. "septicshock") were determined in 8 groups of CBA mice (10 animals per group),using a single ip injection of 1-70 mg/kg LPS (Sigma Chemical Co., St.Louis, Mo.) and recording mortality at 2 hr. intervals from 0 hrs. to 48hrs. The mortality results were evaluated using theLitchfield-Wilcoxon's probit analysis method and a computer programwritten to conduct probit analyses. Having determined these lethal dosevalues, a prophylactic test regimen was conducted using L--Glu--L--Trpat doses of 10 μg/kg and 100 μg/kg ip delivered daily on each of threeconsecutive days prior to the ip LPS injection, with the last of the 3injections being 24 hrs. prior to the LPS challenge. LPS wasadministered in different groups of animals (10 animals/control groupand 11/experimental group) at either the LD₅₀ (58 mg/kg) or LD₉₉ (153mg/kg) dose. The results presented in TABLE 84 show the survival ofanimals at 24 and 48 hrs.

                  TABLE 84                                                        ______________________________________                                        Survival at 24 hrs. and 30 hrs. After an ip                                   Injection of an LD.sub.99 Dose of LPS                                                Time            Dose  No.          Survivors                           Group  (hrs.) Test     (μg/kg)                                                                          Animals                                                                             Survivors                                                                            (%)                                 ______________________________________                                        1      24     Saline    0    10    1      10                                  2      24     Glu--Trp  10   11    4      36                                  3      24     Glu--Trp 100   10    8      80*                                 1      30     Saline    0    10    0      0                                   2      30     Glu--Trp  10   11    1      9                                   3      30     Glu--Trp 100   10    4      40*                                 ______________________________________                                         *p < 0.05                                                                

The results presented in TABLE 84 show that there was a statisticallysignificant difference in the number of survivors at the 24 hr. and 30hr. time points in the animals prophylactically treated with 100 μg/kgof L--Glu--L--Trp.

EXAMPLE 46 Preparation of Analogs, Antagonists and Agonists

The R'-Glu-Trp-R" pharmaceutical compositions of the present inventioncomprise a therapeutically effective amount of a dipeptide having thegeneral formula R'-X-Tryptophan-R" or a pharmaceutically acceptable saltthereof, wherein X is any naturally-occurring amino acid; and apharmaceutically acceptable carrier. Generally, X is glutamine,glutamate, leucine, or isoleucine, R' is a free amino group or amide,and R" is a carboxyl, hydroxyl or carbonyl group. Cyclic and polymericforms of tryptophan-containing dipeptides may also be prepared andtested for activity according to any of the in vitro or in vivo assaysidentified above.

Up to three R'--X--Trp--R" dipeptide subunits may be joined (e.g.,through peptide bonds) to form polymers that can be tested as describedabove. Polymers having the following formulas are synthesized andtested:

    X--Trp--Y--Trp, or

    X--Trp--Y--Trp--Z--Trp,

or pharmaceutically acceptable salts thereof, wherein "X", "Y", and "Z"may be any naturally-occurring amino acids. X, Y, and Z may be the sameor different amino acids. Generally, at least one of X, Y, and Z will beglutamic acid, glutamine, glutamate, or a derivative or analog thereof

R'--X--Trp--R" dipeptides and dipeptide polymers may also cyclized andtested. The cyclic forms so synthesized may have 1, 2, or 3 dipeptidesubunits, e.g., of the general formula

    X--Trp, X--Trp--Y--Trp, or X--Trp--Y--Trp--Z--Trp

or pharmaceutically acceptable salts thereof, wherein X, Y, and Z is anynaturally-occurring amino acid. X, Y, and Z may be the same or differentamino acids. Generally, at least one of X, Y, and Z will be glutamicacid, glutamine, glutamate, or a derivative or analog thereof.

Derivatives of any of the aforementioned dipeptides may also be preparedand tested, e.g., acylated derivatives, amidated derivatives andmethylated derivatives.

Other forms of the dipeptide may also be synthesized and tested,including cyclized monomers such as that representatively depicted inFormula I, below: ##STR1##

wherein R_(a) and R_(b) are, respectively, the alpha-side chains of Gluand Trp, or linear and/or cyclic polymers of EW dipeptide. The linearpolymer is made by conventional peptide synthesis, including Merrifieldsolid-state peptide methodology (described below). The cyclic monomerand polymers is then prepared by cyclizing the linear peptides usingpeptide linking agents, e.g., in dilute solutions.

Easily hydrolyzable polypeptides with multimeric repeats of the EWdipeptide, (e.g., di-dipeptides or tri-dipeptides), can also be preparedand tested for activity according to any of the in vitro or in vivoassays identified above. The hydrolyzable polypeptides include e.g.,anhydrous chlorides or fluorides. When introduced into aqueous solutionthe monomeric EW dipeptides are released from the polypeptides byhydrolysis. Multimers include molecules in which two or more EWcovalently bonded to a common linker, or in which the two or more EWdipeptides are non-covalently linked together through the linker (e.g.,through ion or hydrophobic interactions).

"Molecular mimics" can also be synthesized and tested for activityaccording to any of the in vitro or in vivo assays identified above.,i.e., compounds mimicking and/or exceeding the biological activities ofan R'--Glu--Trp--R" dipeptide. The subject molecular mimics generallyconform to Formula II, below: ##STR2## wherein, R₁ is a neutral polar,neutral nonpolar, or basic residue selected from a functional groupcontaining a halogen atom such as an amine, amide, amido- and the like,or alternatively, R¹ is selected from a functional group containing astraight or branched peptide chain, or straight or branched chainalkyl-, alkoxy-, or cyclic hydrocarbon, such as methyl-, ethyl-,propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, cyclohexyl-, phenoxy-,glycol- and the like, or alternatively, R₁ is selected from ahydrophobic or amphipathic residue such as a glycerol-phosphatide orfatty-acyl chain (e.g., phosphatidyl-ehtanolamine, phosphatidyl-choline,phosphatidyl-inositol, butyryl-, lauryl-, myristyl-, undecyl-, and thelike) or a glycolipid (e.g., cerebroside, ceramide dihexoside and thelike), or one or more hexosyl- residues, (e.g., sucrosyl-, glucosyl-,glucosaminyl-, galactosyl-, galactosaminyl-, lactosyl-, mannosyl-,sorbitolyl-, glycerolyl-, amylosyl- and the like).

R₂ and R₄ are straight or branched alkyl chains selected from methyl-,ethyl-, propyl-, butyl-, pentyl-, hexyl-; isopropyl-, isobutyl-,isopentyl-, isohexyl-, and the like.

R₃ is a negatively charged residue such as a modified carbonyl- residuessuch as carboxylic acids, carboxylic acid amides, acyl-modifiedcarboxylic acids, carbamates, di-alcohols, aldehydes, and the like.

R₅ is an 3-indolyl-like residue such as 3-indolyl-, 3-indolinyl-,9-purinyl-, 1H-indazole-3yl-, 3-isoindolyl-, 3-indolizinyl-,3-isoquinolyl-, 3-quinolyl-, and the like.

R₆ is an electron donor residues such as acetyl-, alkyl-, or modifiedcarbonyl- residues such as carboxylic acids, carboxylic acid amides,acyl-modified carboxylic acids, carbamates, di-alcohols, aldehydes, oralternatively, one or more cyclic or heterocyclic hydrocarbon rings suchas phenyl-, phenoxy-.

Derivatives of R'--Glu--Trp--R" may also be constructed such aslipopeptides having multiple EW residues (e.g., di-, tri-, andtetra-lipopeptides and the like). One illustrative example of aglycerol-phosphatide multimer is provided in Formula III, below, as aphosphatidyl fatty acid of glycerol: ##STR3##

wherein a first EW dipeptide is depicted "aa₁ aa₂ " and a second EWdipeptide "aa₃ aa₄ "; R₁ and R₂ are "non-interfering residues." Thenon-interfering residues R₁ and R₂ may used to stabilize and/or enhancethe biological activity of compound of Formula III. "Non-interferingresidue" as used herein means any chemical residue that when present inposition R₁ or R₆ of Formula II, or position R₁ or R₂ of Formula IIIdoes not interfere with binding of the subject ligand of the respectiveformula to a ligand receptor. For example, non-interfering amino acidresidues or glycosyl residues may be useful in positions R₁ and R₆ ofFormula I (or positions R₁ or R₂ of Formula III) to stabilize or enhancethe biological activity of the synthetic compound.

MATERIALS AND METHODS FOR EXAMPLES 1-45

Staphylococcal strains: Laboratory strains of penicillin-sensitive and-resistant Staphylococci were used in the peritonitis experiments. Theresistant strain was methicillin resistant. Penicillin-resistantstaphylococcal strain ATCC 33593 was obtained from the American TypeCulture Collection, Bethesda, Md.

Antibiotic sensitivity of Staphylococci were determined bydoubling-dilution methods with the aid of the semiautomatic MIC-2000system (Dynatech, USA).

Indices of humoral immunity (e.g., IgG, IgA, IgM) were determined inaccordance with the methods recommended by the World Health Organizationin 1976. Indices of cellular immunity were determined in accordance withaccepted methods published in the Western scientific literature, and insome cases as translated and modified for use with reagents commonlyavailable in the former Soviet Union. Peripheral blood cell differentialcell counts were conducted according to routine methods. Lymphocyteswere isolated from heparinized peripheral blood on Ficoll-Hypaque (δ1.077; Ficoll-Paque, Pharmacia, Switzerland) or Ficoll-Urotrast. Total Tcells were determined by E-rosette formation with ram erythrocytes, andT-helper and T-suppressor subpopulations were determined using indirectimmunofluorescence and monoclonal antibodies OKT4 and OKT8 (Ortho, USA).B-lymphocytes were quantitated by EAC-rosetting and surface expressionof IgG, IgA, or IgM determined using direct immunofluorescencemicroscopy and monospecific antibodies (Sevas, Czechoslovakia,(former)). Immunoglobulin (IgG, IgA, and IgM) and C3 concentrations inperipheral blood were determined using radial immunodiffusion in agaroseand using monospecific antisera (Sevas, supra). Neutrophil phagocyticactivity (% phagocytic cells) and phagocytic index (number of bacteriaper cell) was determined using S. aureus strain Oxford 209P. NaturalKiller (NK) lymphocyte activity was determined at a 50:1 E:T(effector:target) ratio with ³ Uridine labeled K-562 target cells: i.e.,% cytotoxicity (% C.I.) was calculated as follows: % C.I.=1- CPMtest/CPM control×100%!. Production of MIF cytokine was measured in vitrousing a modification of the method described in "Evaluation of theBody's Immune Status in Therapeutic Institutions of the Soviet Army andNavy," Ministry of Defense, U.S.S.R., Center of Military Medicine, 1987(L. A. Kozhemia, ed). MIF production was measured following stimulationof Ficoll-Hypaque-purified lymphocytes with mitogens, i.e., PHA,(phytohemagglutinin, Serva, West Germany (former); ConA,(Concanavalin-A, Serva), and antigens, i.e., hemolytic staphylococcalallergen (HSA; Kazanskii, SRI of Epidemiology and Microbiology,U.S.S.R.).

Peripheral Blood and Serum Samples: Patient and animal samples ofperipheral blood were collected into heparin and serum was obtained fromseparate blood samples collected and clotted in glass tubes.

Thymus, Lymph Node, Bone Marrow and Spleen Samples: Thymus, leftparatracheal lymph node, and spleen were surgically removed, weighed,and homogenized in a glass Dounce homogenizer in Medium 199. Tissuefragments were removed by filtration through a chaperon. Bone marrowcell suspensions were prepared by irrigating the bone marrow canal ofthe pelvis with Medium 199 and homogenizing the irrigated solution.Suspensions from the different respective sources were purified bycentrifuging the different cell samples on different aliquots of δ 1.077Ficoll-Urotrast for 40 minutes at 1500-1800 rpm. The lymphocyteinterfaces from the gradients were collected into a siliconized testtube and washed in Medium 199. Residual red cells in the lymphocytesuspensions were lysed using 3% acetic acid in the presence of 0.05%Hessian-violet as a viability marker. Cell concentrations weredetermined microscopically using a Goryaev counting chamber. Cellconcentrations were adjusted to 2.5×10⁶ /mL.

Differential Cell Counts: Differential cell counts were performed bylysing erythrocytes in peripheral blood with 3% acetic acid, washing,and preparing smears of cells on a slide. After air-drying the cells inthe smear were stained using the method of Romanovsky-Geimsa.Percentages of the different cell types were determined by counting aminimum of 100 cells in each sample.

Lymphocyte Preparations Lymphocytes were prepared by density gradientcentrifugation on δ 1.077 Ficoll-Urotrast (Pharmacia).

E-RFC: Ram erythrocytes (E) were washed 4× in saline (2000 rpm/10 min.)and suspended at a final concentration of 1% in Medium 199. Fordeterminations of the percentage of E-rosette forming T-lymphocytes(E-RFC) in a sample, 0.1 mL of a prepared lymphocyte suspension (2.5×10⁶/ml) was incubated with 0.1 mL of the 1% ram erythrocyte suspension at37° C for 10 min., centrifuged at 800-1000 rpm for 5 min., maintained at4° C. for 2 hrs., and finally resuspended and counted in a Goryaevchamber. Activated T-lymphocytes (i.e., having increased E-RFC capacity)were determined by mixing the test lymphocyte suspension with E,immediately centrifuging for 5 min. at 800-1000 rpm, resuspending thecells and counting the rosettes formed in a Goryaev chamber.

EA-RFC: Antibody-coated rabbit erythrocytes were used for determinationsof EA-RFC (i.e., "activated" T-lymphocytes) according to the method ofStadecker, M. J., et al., J. Immunol. 11(6):1834-1837 (1973).Erythrocytes were prepared, incubated with an optimal dilution ofanti-rabbit IgG, washed, and prepared as a 1% cell suspension in amanner analogous to those above (E-RFC, supra). EA-RFC assays wereperformed in a manner analogous to the description above, i.e., E-RFC,supra.

EAC-RFC: Determinations of EAC-RFC (i.e., B-lymphocytes) were performedaccording to the method described by Bianco, C., et al., J. Exp. Med.132(4):702-720 (1970).

CD3⁺, CD4⁺, CD19⁺, and CD8⁺ Lymphocytes: To enumerate populations oflymphocytes, indirect immunofluorescence, (using Ortho OKT monoclonalantibodies according to the manufacturers instructions), and a directfluorescence microscope equipped with phase contrast optics, employed.Cells having "ring" or "point" luminescence were counted. Diffusestaining was not counted. Determinations of the percentages of T- andB-lymphocytes were made by counting a minimum of 200-300 cells.

Blastogenesis with PHA and Con-A: Lymphocyte blastogenic responsivenessto mitogens was evaluated using the method of Bach, J. F., et al., Proc.Ann. Leukocyte Culture Conf. p. 271-283 (1971)).

LMIR: Lymphocyte migration inhibition factor production was determinedaccording to a modification of Bendixsen, G. et al. 1980 by: i) addingConcanavalin-A to a sample of a patient's heparinized blood (25 units/mlheparin) to achieve a final concentration of 80 μg/ml Con-A; ii)collecting 200 μl of the Con-A-heparinized blood into a smooth boreheparinized glass capillary tube; iii) sealing one end of the capillarytube with paraffin or plastylene; iv) centrifuging the capillary for 5minutes at 1500-2000 rpm; v) incubating the capillary tube for 18-24hrs. at 37° C. in tissue culture medium in a vertical orientation; vi)determining the diameter of the zone of leukocyte migration at theborder of the erythrocyte mass using an ocular micrometer; and, vii)subtracting from the recorded values the migration area in controlsamples (i.e., packed blood lacking Con-A). The calculation of LMIR %was as follows:

Migration inhibition (LMIR) %=diameter of the migration zone in thepresence

of Con-A/ diameter of the migration zone in the absence of Con-A X 100%.Three capillary tubes were used for each determination, and the mean ofthe values recorded in the determinations +/-S.D. are presented above.

Lysosomal Cation Test: The lysosomal cation test (LCT) characterizesoxygen-independent metabolic processes in neutrophils. Neutrophildegranulation results in release of proteases (and cationic proteins),increased permeability of neutrophil membranes, increased oxygenconsumption, activation of the hexose monophosphate shunt, and formationof hydrogen peroxide in lysosomes. Neutrophil cationic proteins are ameasure the activation state of neutrophils and levels of these proteinsare correlated with the level of non-specific resistance to infection inan animal (or man).

To determine neutrophil cationic proteins, briefly, blood smears wereair dried, stained for 20 minutes in a buffered methanol-fast greensolution, washed in distilled water, and stained for an additional 30minutes with Azure 11. The percentage of cells containing green-coloredgranules was determined by microscopically counting a minimum of 100-200of the cells in the smears, and cation protein concentration wasrecorded as the mean cytotoxic coefficient (MCC) according to a modifiedformula of Astold & Berg.

Immunoglobulins and Acute Phase Reactant Protein Levels: Concentrationsof IgG; IgM and IgA and acute phase reactant proteins (e.g., α₂-macroglobulin, orosomucoid, prealbumin, etc.) in serum were determinedby radial immunodiffusion in agarose according to the method of Mancini(1965).

Bacterial Challenge: To insure a uniform inoculum of bacteria inEXAMPLES 32-34, above, suspensions were shaken at 30° C. for 2 hours ona rotary platform shaker in 1.5× brain heart infusion broth. Dispersedsingle cells were collected by centrifugation 3000 rpm/20 min. andresuspended in 1X brain heart infusion broth at a cell number equivalentto 10 times the LD₅₀ dose of bacteria, and then 5% sterile mucin wasadded to stabilize the inoculum. Animals were injected ip with 10 timesthe LD₅₀ number of bacteria suspended in the brain-heart infusion brothcontaining the 5% mucin.

Therapeutic Treatment: In EXAMPLES 32-34, above, test substance (i.e.,L--Glu--L--Trp alone or in combination with antibiotic, or antibioticalone) was injected sc, ip, po one hour after the bacterial challenge.

Prophylactic Treatment: In EXAMPLES 32-34, above, test substance (i.e.,L--Glu--L--Trp alone or in combination with antibiotic, or antibioticalone) was injected daily ip for the three days immediately prior to thebacterial challenge.

Viral Assays: In EXAMPLE 38, SC-1 cells were originally derived frommurine embryos, and XC cells (a rat tumor cell line induced by RousSarcoma Virus) were obtained from the ATCC in Rockville, Md.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method for treating an autoimmune disease in ananimal comprising the step of administering to the animal an effectiveamount of L--Glu--L--Trp or a salt thereof.
 2. The method of claim 1wherein the animal is a human.
 3. The method of claim 1 wherein theanimal is non-human.
 4. The method of claim 2 wherein the amount is 0.1μg/kg to 1 μg/kg body mass of the human.
 5. The method of claim 2wherein the amount is 1 μg/kg body mass of the human.
 6. The method ofclaim 2 wherein the step comprises administering 500 μg ofL--Glu--L--Trp or a salt thereof over several doses.
 7. The method ofclaim 2 wherein the step comprises administering L--Glu--L--Trp or asalt thereof by intravenous injection.
 8. The method of claim 2 whereinthe step comprises administering L--Glu--L--Trp or a salt thereofintranasally.
 9. The method of claim 2 wherein the step comprisesadministering L--Glu--L--Trp or a salt thereof intramuscularly.
 10. Themethod of claim 2 wherein the step comprises administeringL--Glu--L--Trp or a salt thereof in the form of a tablet, a solution, adrop, an ointment or a suppositorium.
 11. The method of claim 2 whereinthe step comprises administering L--Glu--L--Trp or a salt thereof in theform of an injectable solution further comprising novocain, Ringer'ssolution, or glucose.
 12. The method of claim 2 wherein the autoimmunedisease is rheumatoid arthritis.